Download Modes of anaerobic respiration catalysed by

Modes of anaerobic respiration catalysed by members of the
class Dehalococcoidia
Vorgelegt von
Dipl. Biologin Myriel Cooper
geb. in Hamburg, Deutschland
von der Fakultät III – Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
Dr.-rer. nat.
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Jens Kurreck
1. Gutachter: Prof. Dr. Peter Neubauer
2. Gutachter: PD Dr. Lorenz Adrian
3. Gutachterin: Dr. habil. Corinne Petit-Biderre
Tag der wissenschaftlichen Aussprache: 12. Oktober 2015
Berlin 2016
Erklärung zur Dissertation
Ehrenwörtliche Erklärung zu der Dissertation mit dem Titel:
“Modes of anaerobic respiration catalysed by members of the class Dehalococcoidia”
Hiermit erkläre ich, dass ich die vorliegende Dissertation selbstständig angefertigt und keine anderen
als die von mir angegebenen Quellen und Hilfsmittel verwendet habe. Alle wörtlich oder inhaltlich
übernommenen Stellen habe ich als solche gekennzeichnet. Wörtlich oder inhaltlich Stellen, die aus
meinen persönlichen Publikationen stammen, wurden nicht zusätzlich gekennzeichnet. Ich erkläre
weiterhin, dass die Dissertation bisher nicht in dieser oder anderer Form in einem anderen
Prüfungsverfahren vorgelegen hat und, dass diesem Promotionsverfahren keine endgültig
gescheiterten Promotionsverfahren vorausgegangen sind.
Ort, Datum
Unterschrift
Acknowledgement
Acknowledgement
The present work was performed from April 2010–April 2014 at the Department of Isotope
Biogeochemistry, Helmholtz Centre for Environmental Research Leipzig (UFZ). The PhD thesis was
carried out in the research group of PD Dr. Lorenz Adrian. The research project was funded by the
European Research Council (Project “Microflex” - Proposal Nr. 202903).
Firstly, I express my sincere gratitude to Dr. Lorenz Adrian for giving me the opportunity to work and
prepare my thesis in this exciting research area and fantastic working group. I also thank him for the
supervision of my thesis, the constant support, the many inspiring discussions, and for sharing his
knowledge and passion for the microbial world.
I would like to thank Prof. Dr. Peter Neubauer for the external supervision and review of my
dissertation, Dr. habil. Corinne Petit-Biderre for reviewing my dissertation and Prof. Dr. Jens Kurreck
to be head of the dissertation committee.
Dr. Anke Wagner I thank for sharing her great knowledge of the “Dehalococcoides-world” with me,
for her supervision and the constant support, the productive cooperation and her friendship during all
of the years we have been working together. My colleague Dr. Kenneth Wasmund I thank for the great
cooperative work, his invaluable suggestions and advice and for his friendship. Further, I want to
thank Camelia Algora, Anja Kublik, Chao Yang and Darja Deobald for countless interesting and
helpful discussions, their support and their friendship. I thank Benjamin Scheer and Bernd Krostitz for
the technical support and continuous help in the laboratory. I thank Benjamin Scheer furthermore for
organizing so many great activities with our lab group. My student Jessica Zhang I would like to thank
for her endurance during difficult experiments.
Dominik Wondrousch and Prof. Dr. Gerrit Schüürmann from the Department for Ecological
Chemistry of the UFZ I would like to thank for their important contributions from the field of
theoretical chemistry and the fruitful cooperation. I would like to thank all co-authors for their work
and contributions.
PD Dr. Hans Richnow and all members of the Biogeochemistry Department I thank for their support,
helpful discussions and advice. Prof. Dr. Jens Kurreck and his working group I thank for hosting me in
their laboratories during my stay at the Technical University of Berlin and the wonderful time I could
spend with them.
Finally, I thank my family and Martin Ferreux for their love and their support during the last years and
for being part of my life.
i
Summary
Summary
The bacterial class Dehalococcoidia, phylum Chloroflexi, encompasses several cultivated strains and
many so far uncultivated bacteria, known from molecular detection of their 16S rRNA gene sequences
in marine sediments. All cultivated representatives exclusively respire with halogenated compounds as
terminal electron acceptor in a process termed organohalide respiration. However, it is not known
whether organohalide respiration can explain the abundance and ubiquitous distribution of
Dehalococcoidia in marine pristine sediments.
In the current work, electron acceptors for respiration of Dehalococcoidia were investigated with
the model organism Dehalococcoides mccartyi strain CBDB1 to gain insight into which properties of
halogenated compounds are required to serve as electron acceptor of Dehalococcoidia and might in
part explain their abundance at marine sites. For this, a microtiter plate-based assay was established to
measure reductive dehalogenase activity  the key enzyme in organohalide respiration. D. mccartyi
strain CBDB1 dehalogenated chlorinated benzonitriles, chlorinated anilines and brominated phenols,
benzenes, pyridines, furoic and benzoic acids and specific activities of 4.5 to 241 nkat mg-1 protein
were determined. In cultivation experiments, growth yields of 0.1 x 1014 to 2.3 x 1014 cells mol-1
halogen released were obtained with strain CBDB1. Brominated electron acceptors were generally
dehalogenated to a further extent than their chlorinated equivalents and showed higher specific activity
rates in resting cell assays. Results of shotgun proteomics and activity assays suggest that the same
reductive dehalogenases are involved in the dehalogenation of brominated and chlorinated benzenes,
indicating chemical properties influence reductive dehalogenation patterns of strain CBDB1. The
correlation of density functional theory calculations with microbial and biochemical experiments
revealed that functional groups decreasing electron density at the halogen and not at the
halogen-substituted carbon enhance reductive dehalogenation. The most positive halogen partial
charge predicted the regioselective dehalogenation catalysed by strain CBDB1 for up to 96% of all
evaluated molecules. These findings suggest a cobalamin–halogen interaction during reductive
dehalogenation which stands in contrast to previous models of reductive dehalogenation. A halogen–
cobalamin interaction could in part explain the broad electron acceptor diversity of organohaliderespiring Dehalococcoidia suggesting that a multitude of natural organohalogens – including
brominated aromatics – could serve as natural halogenated electron acceptor for respiration and
growth of Dehalococcoidia in marine sediments. In addition, the established prediction systems may
allow for improved fate prediction of halogenated compounds from anthropogenic or natural sources
in the environment. Reductive dehalogenation of brominated aromatics was also shown to occur in
marine deep-sea sediment microcosms with activity assays, inhibition studies and cultivation
experiments although no Dehalococcoidia were detected. However, the conducted experiments
demonstrated that similar dehalogenation patterns observed with model organism strain CBDB1 occur
in non-contaminated deep-sea sediments. To investigate the potential of Dehalococcoidia to respire
ii
Summary
non-halogenated
electron
acceptors,
the
genetic
information
from
the
single
amplified
Dehalococcoidia genome SAG-C11 was used to design primers for a newly identified class of
dissimilatory sulphite reduction genes (dsr) in Chloroflexi. The primers were used to study sediment
samples from different locations and depths for the presence of Chloroflexi-related dsr genes. Several
dsr genes located in a similar genetic context as observed in SAG-C11 and affiliating with dsr genes
from SAG-C11 in phylogenetic analyses were detected in sediments from Aarhus, the Baffin Bay and
in tidal flat sediments of the Wadden Sea. The obtained dsrAB genes shared 71–100% nucleotide
sequence identities with dsrAB of SAG-C11 suggesting that dissimilatory sulphite reduction is a more
widespread mode of respiration in the class Dehalococcoidia and could contribute together with
organohalide respiration of brominated natural electron acceptors to the abundance and ubiquitous
presence of Dehalococcoidia in marine sediments.
iii
Zusammenfassung
Zusammenfassung
Die vorliegende Arbeit beschreibt Formen der anaeroben Atmung der bakteriellen Klasse
Dehalococcoidia,
Abteilung
Chloroflexi,
welche
zu
den
häufigsten
und
abundantesten
Bakteriengruppen mariner Sedimente gehört und deren kultivierten Vertreter aus terrestrischen
Habitaten ausnahmslos auf eine Atmung mit halogenierten organischen Verbindungen (Organohalidatmung) angewiesen sind. Kultivierung und Mikrotiterplatten-basierte Aktivitätstests mit strukturell
unterschiedlichen Elektronenakzeptoren zeigten, dass Dehalococcoidia Modellorganismus Stamm
CBDB1 chlorierte Benzonitrile, chlorierte Aniline und bromierte Phenole, Benzole, Pyridine, Furonund Benzoesäuren dehalogenierte. Wachstumsausbeuten von 0,1 x 1014 bis 2,3 x 1014 Zellen pro Mol
freigesetztem Halogen Anion und spezifische Aktivitäten von 4,5 bis 241 nkat pro mg Protein wurden
gemessen. Bromierte Aromaten wurden weitergehend und mit höheren spezifischen Aktivitäten
dehalogeniert als ihre chlorierten Äquivalente. Aktivitätstests und Shot-Gun Proteomik deuteten
darauf hin, dass die gleichen reduktiven Dehalogenasen in die Umsetzung der getesteten bromierten
und chlorierten Benzole involviert sind. Bromierte aromatische Verbindungen, die in marine Habitaten
vorkommen, könnten somit als
natürliche Elektronenakzeptoren für Organohalid-atmende
Dehalococcoidia in marinen Sedimenten dienen. Eine reduktive Dehalogenierung bromierter
Verbindungen wurde auch in Aktivitätstests mit Zellen aus marinen Sediment-Mikrokosmen und
durch Inhibitionsstudien nachgewiesen. Obwohl keine Chloroflexi in den marinen SedimentMikrokosmen detektiert werden konnten, zeigten die Experimente, dass reduktive Dehalogenierung
auch in marinen nicht-kontaminierten Tiefseesedimenten vorkommen kann. Die Korrelation
mikrobiologischer und biochemischer Versuche mit Dichtefunktionaltheorie-basierten Berechnungen
verschiedener Partialladungs-Modelle zeigte, dass die regioselektive Dehalogenierung durch Stamm
CBDB1 mit Hilfe der positivsten Halogen-Partialladung für 96% der untersuchten Moleküle
vorausgesagt werden konnte. Außerdem konnte gezeigt werden, dass funktionelle Gruppen, die die
Elektronendichte am Halogenatom verringern, die reduktive Dehalogenierung unterstützen. Dies weist
auf eine Cobalamin–Halogen Interaktion während der reduktiven Dehalogenierung hin und steht im
Gegensatz zu bisherigen Modellen der reduktiven Dehalogenierung. Eine Cobalamin–Halogen
Interaktion könnte dazu beitragen, das breite Elektronenakzeptoren Spektrum Organohalid-atmender
Dehalococcoidia zu erklären und könnte Organohalid-atmenden Dehalococcoidia in marinen
Sedimenten erlauben, verschiedenste halogenierte Verbindungen wie z.B. bromierte komplexe
Aromaten für die Atmung zu nutzen. In einem weiteren Teil dieser Dissertation wurde mit
molekularbiologischen Methoden das Potential von Dehalococcoidia untersucht, nicht-halogenierte
Elektronenakzeptoren zu nutzen. Dies könnte die Stratifikation von Dehalococcoidia Subgruppen
entlang geochemischer Gradienten erklären. Dazu wurden mit Hilfe der Genominformation einer
Dehalococcoidia-Einzelzelle („SAG-C11“) PCR-Primer entwickelt, die spezifisch dsr Gene in
Dehalococcoidia amplifizierten. Mit diesen Primern wurden das Vorkommen und die Diversität der
iv
Zusammenfassung
dissimilatorischen Sulfitreduktase (dsr) in Dehalococcoidia in Sedimenten verschiedener Orte und
Tiefen untersucht. Unterschiedliche dsr Gene, die sich in einem ähnlichen genetischen Kontext wie in
SAG-C11 befanden und in phylogenetischen Analysen mit dsr Sequenzen aus SAG-C11 gruppierten,
wurden in Sedimenten der Aarhusbucht, der Baffinbucht und in Wattsedimenten der Nordsee
nachgewiesen. Nukleotidsequenzähnlichkeiten von 71–100% der aus marinen Sedimenten
amplifizierten dsrAB-Sequenzen zu den dsrAB-Sequenzen aus SAG-C11 weisen darauf hin, dass
dissimilatorische Sulfitreduktion ein verbreiteter Atmungsprozess in Dehalococcoidia ist und
zusammen mit der Organohalidatmung bromierter Verbindungen zu einer Erklärung der ubiquitären
Verbreitung von Dehalococcoidia in marine Sedimenten in unterschiedlichen biogeochemischen
Zonen beitragen kann.
v
List of publications and contributions
List of publications and author contributions
Publication 1 (*shared first authorship):
Anke Wagner*, Myriel Cooper*, Sara Ferdi, Jana Seifert, Lorenz Adrian: “Growth of
Dehalococcoides mccartyi strain CBDB1 by reductive dehalogenation of brominated
benzenes to benzene”, Environmental Science & Technology, 2012, 46 (16), 8960–8968,
DOI: 10.1021/es3003519
Contributions: Anke Wagner, Myriel Cooper and Lorenz Adrian developed the concept of the
study. Cultivation studies of strain CBDB1 were done by Anke Wagner and Sara Ferdi.
Cultivation for activity assays was done by Myriel Cooper, Anke Wagner and Bernd Krostitz.
Myriel Cooper established and applied the activity assay and evaluated the data from activity
measurements. Shotgun proteomics of bromobenzene cultures was done by Jana Seifert and
data were evaluated by Myriel Cooper and Lorenz Adrian. The manuscript was drafted by
Anke Wagner and Myriel Cooper and revised by Lorenz Adrian.
Publication 2:
Myriel Cooper, Anke Wagner, Dominik Wondrousch, Frank Sonntag, Andrei Sonnabend,
Martin Brehm, Gerrit Schüürmann, and Lorenz Adrian: “Anaerobic microbial transformation
of halogenated aromatics and fate prediction using electron density modelling”,
Environmental
Science
&
Technology,
2015,
49
(10),
6018-6028,
DOI:
10.1021/acs.est.5b00303.
Contributions: Myriel Cooper, Anke Wagner and Lorenz Adrian developed the concept for
the study. Dominik Wondrousch calculated electron density distributions, including sigma
and pi partial charges. Martin Brehm calculated the electron density distributions in solvents.
The evaluation of electron density distributions for dehalogenation pathway prediction was
done by Myriel Cooper and revised by Anke Wagner. The model for the newly proposed
reaction mechanism was developed by Anke Wagner, Myriel Cooper, Gerrit Schüürmann and
Lorenz Adrian. Cultivation of strain CBDB1 with chlorinated anilines, 2-chloro-6fluorobenzonitrile, chlorinated pyridines, brominated furoic and benzoic acids was done by
Myriel Cooper. Frank Sonntag cultivated strain CBDB1 with chlorinated benzonitriles and
Andrei Sonnabend with 3-bromo-2-chloropyridine and chlorinated thiophenes. Data
evaluation was done by Myriel Cooper and Anke Wagner. Activity assays and data evaluation
were conducted by Myriel Cooper. The manuscript was drafted by Myriel Cooper and Anke
Wagner, Gerrit Schüürmann and Lorenz Adrian revised the manuscript.
vi
List of publications and contributions
Publication 3 (in preparation, *shared first authorship):
Kenneth Wasmund*, Myriel Cooper*, Lars Schreiber, Karen G. Lloyd, Brett Baker, Dorthe
G. Petersen, Andreas Schramm, Ramunas Stepanauskas, Richard Reinhardt, Bo Barker
Jørgensen, Alexander Loy, Lorenz Adrian: Single cell genome sequencing and targeted gene
amplifications implicate members of the class Dehalococcoidia (phylum Chloroflexi) in
sulfur cycling.
Contributions: Kenneth Wasmund and Lorenz Adrian developed the concept of the study.
Lars Schreiber, Karen Lloyd, Brett Baker, Dorthe G. Petersen, Andreas Schramm and Bo
Barker Jørgensen were involved in the sampling, planning and initiating single cell isolations.
Single cell isolations were done by Ramunas Stepanauskas. Sequencing was done by Richard
Reinhardt. Lars Schreiber and Kenneth Wasmund assembled the genome, Kenneth Wasmund
analysed the assembly and annotated the genes. Pathways integration was done by Kenneth
Wasmund and Lorenz Adrian. Alexander Loy contributed to annotation and analysis of dsrgenes. Myriel Cooper and Kenneth Wasmund developed the concept for targeted gene
amplification. For this, Myriel Cooper and Lars Schreiber sampled sediments at Aarhus Bay.
Experiments for targeted gene amplification and data evaluation were done by Myriel Cooper.
The manuscript was written by Kenneth Wasmund, Myriel Cooper and Lorenz Adrian.
vii
Table of contents
Abbreviations
16S rRNA 16S ribosomal ribonucleic acid
APR Adenylyl‐sulphate‐reductase
Blast Basic Local Alignment Search Tool
BLASTn Standard Nucleotide BLAST
CbrA Chlorobenzene reductive dehalogenase
CID Collision‐induced dissociation
cmbsf centimetres below sea floor
Co(I) Cob(I)alamin
Co(II) Cob(II)alamin
Co(III) Cob(III)alamin
CobA/HemD uroporphyrinogen III methyl transferase/synthase
CPh Chlorophenol
CPhate Chlorophenolate
DBB Dibromobenzene
DCB Dichlorobenzene
DCPh Dichlorophenol
DCPhate Dichlorophenolate
DMSO Dimethyl sulfoxide
dNTP Nucleoside triphosphate
drsAB Dissimilatory sulphite reductase subunit A and B genes
Dsr Dissimilatory sulphite reductase
emPAI Exponentially modified protein abundance index
FID Flame ionization detector
fw Forward
GC Gas chromatograph
GluRS Porphobilinogen deaminase, glutamyl‐
tRNA reductase
HCB Hexachlorobenzene
HPLC High performance liquid chromatography
LB Lysogeny broth
LC-ESI-MS/MS Liquid chromatography
electrospray ionization mass
spectrometry/ mass spectrometry
MarR Multiple antibiotic resistance regulator
MBB Monobromobenzene
MbrA Chloroethene reductive dehalogenase
MCB Monochlorobenzene
MV Methyl viologen
NCBI National Center for Biotechnology Information
nr non‐reduntant
ODP Ocean drilling program
PCE Tetrachloroethene
PceA Tetrachlorethene reductive dehalogenase
PeCB Pentachlorobenzene
PeCPh Pentachlorophenol
PeCPhate Pentachlorophenolate
rdh Reductive dehalogenase homologous
rdhA Reductive dehalogenase homologous subunit A gene
RdhA Reductive dehalogenase subunit A
rdhB Reductive dehalogenase homologous subunit B gene
rv Reverse
SOC Super optimal broth
sp. Species
TAE Tris‐Acetate EDTA buffer
TAT Twin arginine transport
TBB Tribromobenzene
TCB Trichlorobenzene
TCE Trichloroethene
TceA Trichloroethene reductive dehalogenase
TCPh Trichlorophenol
TCPhate Trichlorophenolate
TE-buffer Tris‐EDTA buffer
TeCB Tetrachlorobenzene
TeCPh Tetrachlorophenol
TeCPhate Tetrachlorophenolate
UPLC Ultra performance liquid chromatography
VcrA Vinyl chloride reductive dehalogenase
w/v Weight per volume
X-Gal 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐
galactopyranoside
viii
Table of contents
Table of contents
1 INTRODUCTION ................................................................................................................................ 1 1.1 THE CLASS DEHALOCOCCOIDIA .......................................................................................................................... 1 1.1.1 Isolated and cultivated strains of the class Dehalococcoidia ............................................................ 1 1.1.2 Dehalococcoidia in the marine subsurface ....................................................................................... 2 1.2 ORGANOHALIDE RESPIRATION ........................................................................................................................... 5 1.3 ELECTRON ACCEPTORS FOR DEHALOCOCCOIDIA .................................................................................................... 8 1.3.1 Electron acceptors of cultivated Dehalococcoidia............................................................................. 8 1.3.2 Natural organohalogens as potential electron acceptors in marine sediments ............................... 9 1.3.3 Non‐halogenated electron acceptors for Dehalococcoidia in marine sediments ............................ 10 1.4 GOAL OF THIS WORK ..................................................................................................................................... 12 2 MATERIAL AND METHODS ............................................................................................................. 13 2.1 CHEMICALS AND GASES .................................................................................................................................. 13 2.2 BACTERIAL CULTURES AND INOCULA ................................................................................................................. 13 2.2.1 Cultures ........................................................................................................................................... 13 2.2.2 Sediment samples ........................................................................................................................... 14 2.3 CULTIVATION ............................................................................................................................................... 15 2.3.1 Medium preparation for anaerobic cultivation ............................................................................... 15 2.3.2 Cultivation of E. coli ........................................................................................................................ 18 2.3.3 Direct microscopic counting ............................................................................................................ 18 2.4 PREPARATION OF WHOLE CELLS SUSPENSIONS AND DETACHMENT OF CELLS FROM SEDIMENTS ...................................... 19 2.4.1 Rotary evaporator ........................................................................................................................... 19 2.4.2 Cell concentration via Minisart® filter ............................................................................................. 19 2.4.3 Cell concentration via centrifugation .............................................................................................. 19 2.4.4 Density centrifugation using Nycodenz® ......................................................................................... 19 2.5 RESTING‐CELL ACTIVITY ASSAY ......................................................................................................................... 20 2.5.1 Preparation of solutions and cell suspensions for the activity assay .............................................. 21 2.5.2 Gas chromatography‐based activity assay ..................................................................................... 21 2.5.3 Photometer‐based microtiter plate activity assay .......................................................................... 22 2.5.4 Heat inactivation of cells for activity assay ..................................................................................... 23 2.5.5 Reversible inhibition of reductive dehalogenases ........................................................................... 23 2.5.6 Oxygen sensitivity tests with marine cultures and activity assay ................................................... 24 2.5.7 Activity assays with cells from sediments ....................................................................................... 24 2.6 ANALYTICAL METHODS .................................................................................................................................. 25 2.6.1 Gas chromatography ...................................................................................................................... 25 2.6.2 Ion chromatography ....................................................................................................................... 26 2.6.3 NanoLC‐ESI‐MS/MS (LTQ‐Orbitrap) analysis ................................................................................... 26 2.7 MOLECULAR METHODS .................................................................................................................................. 27 2.7.1 Chloroform‐based DNA extraction .................................................................................................. 27 2.7.2 DNA extraction with the FastDNATM Spin Kit for Soil ....................................................................... 28 2.7.3 Whole genome amplification .......................................................................................................... 28 2.7.4 Determination of DNA concentration and purity ............................................................................ 28 2.7.5 Primer combinations and expected products .................................................................................. 28 2.7.6 Polymerase chain reaction .............................................................................................................. 30 2.7.7 Agarose gel electrophoresis ............................................................................................................ 32 2.7.8 Elution of DNA from agarose gels ................................................................................................... 32 2.7.9 DNA and plasmid preparation and purification .............................................................................. 32 2.7.10 Addition of an poly adenosine tail .................................................................................................. 33 2.7.11 TA cloning ....................................................................................................................................... 33 2.7.12 Blunt end cloning ............................................................................................................................ 33 2.7.13 Transformation ............................................................................................................................... 33 2.7.14 Colony PCR ...................................................................................................................................... 33 2.7.15 Screening and sequencing of inserts ............................................................................................... 34 2.8 BIOINFORMATIC TOOLS .................................................................................................................................. 35 ix
Table of contents
2.9 ELECTRON DENSITY DISTRIBUTIONS FOR HALOGENATED AROMATICS ........................................................................ 35 3 RESULTS ......................................................................................................................................... 36 3.1 RESPIRATION AND GROWTH OF DEHALOCOCCOIDES MCCARTYI STRAIN CBDB1 WITH HALOGENATED AROMATIC AND HETEROAROMATIC ELECTRON ACCEPTORS .......................................................................................................... 36 3.1.1 Chlorinated anilines as electron acceptor ....................................................................................... 36 3.1.2 2,3‐Dichloronitrobenzene as electron acceptor .............................................................................. 37 3.1.3 Dehalogenation of and growth with 2‐chloro‐6‐fluorobenzonitrile ................................................ 38 3.1.4 Chlorinated pyridines as electron acceptor ..................................................................................... 39 3.1.5 Brominated phenols as electron acceptor ...................................................................................... 40 3.1.6 Brominated furoic acids as electron acceptor ................................................................................. 41 3.1.7 Brominated benzoic acids as electron acceptor .............................................................................. 43 3.2 SET‐UP OF A PHOTOMETRIC MICROTITER PLATE‐BASED RESTING CELL ACTIVITY ASSAY ................................................. 45 3.2.1 Material choice for a 96‐well microtiter plate for dehalogenase activity measurement ................ 45 3.2.2 Analysis of background oxidation of methyl viologen in the microtiter plate activity assay .......... 47 3.2.1 A total number of 560 cells are required for activity detection in 96‐well microtiter plate tests ... 51 3.2.2 Microtiter plate‐analysed activities are comparable to GC‐FID‐analysed activities ....................... 51 3.3 ANALYSIS OF ENZYME ACTIVITY OF D. MCCARTYI STRAIN CBDB1 ........................................................................... 52 3.3.1 Specific activities of resting cells of D. mccartyi strain CBDB1 with halogenated compounds ....... 52 3.3.2 Attempts to improve sensitivity of the activity assay ..................................................................... 54 3.3.3 Analysis of the influence of the electron acceptor used for cultivation onto resting cell activity and reductive dehalogenase expression using brominated benzenes ................................................... 54 3.4 POTENTIAL FOR ORGANOHALIDE RESPIRATION IN DEEP MARINE NON‐CONTAMINATED SEDIMENTS ................................ 57 3.4.1 Dehalogenation activity with brominated electron acceptors with cells of marine microcosms from the coast off Chile ........................................................................................................................... 57 3.4.2 Characterization of dehalogenation activity in marine sediments ................................................. 58 3.4.3 Dehalogenation activity in Chloroflexi‐containing sediments ......................................................... 60 3.5 CHEMICAL PROPERTIES OF HALOGENATED AROMATIC COMPOUNDS INFLUENCE REDUCTIVE DEHALOGENATION PATTERNS .. 61 3.5.1 The most positive halogen atomic net charge predicts regioselective dehalogenation by strain CBDB1 ............................................................................................................................................. 62 3.5.2 Sigma NBO halogen partial charges predict regioselective dehalogenation .................................. 65 3.5.3 Analysis of the influence of different secondary substituents onto the halogen atomic net charges
65 3.5.4 The influence of chemical properties of heterocycles onto reductive dehalogenation ................... 67 3.5.5 The role of the halogen type in reductive dehalogenation ............................................................. 67 3.5.6 Correlation of the most positive halogen partial charge with resting cell activity ......................... 67 3.6 NON‐HALOGENATED COMPOUNDS AS POTENTIAL RESPIRATORY ELECTRON ACCEPTORS FOR DEHALOCOCCOIDIA .............. 68 3.6.1 Selection of sediment cores and cell extraction .............................................................................. 70 3.6.2 Selection of fragments for analysis ................................................................................................. 71 3.6.3 Linking dissimilatory sulphite reductase genes with Dehalococcoidia ............................................ 73 4 DISCUSSION ................................................................................................................................... 80 4.1 STRATEGIES TO INVESTIGATE ELECTRON ACCEPTORS USED BY BACTERIA OF THE CLASS DEHALOCOCCOIDIA ...................... 81 4.1.1 Cultivation of the model organism D. mccartyi strain CBDB1 ......................................................... 81 4.1.2 Activity measurements in a microtiter plate‐based assay for identification of new halogenated electron acceptors ........................................................................................................................... 82 4.1.3 Analysis of reductive dehalogenase expression as a strategy to gain insights into enzyme specificity ........................................................................................................................................ 84 4.1.4 Evaluation of density functional theory‐based data as a strategy to elucidate chemical parameters influencing reductive dehalogenation ............................................................................................. 85 4.1.5 Analysis of Dehalococcoidia genes associated with respiratory functions in marine sediments .... 86 4.2 THE ELECTRON ACCEPTOR DIVERSITY OF D. MCCARTYI STRAIN CBDB1 AND MARINE DEHALOGENATING MICROORGANISMS 88 4.2.1 Growth of D. mccartyi strain CBDB1 with halogenated electron acceptors ................................... 88 4.2.2 Dehalogenation pathways of halogenated electron acceptors catalysed by strain CBDB1............ 90 4.2.3 Specific activities of strain CBDB1 with halogenated electron acceptors ....................................... 93 4.2.4 Activity assays and shotgun proteomics show the same reductive dehalogenases transform several halogenated compounds .................................................................................................... 94 x
Table of contents
4.2.5 Dehalogenation activity in marine sediments and sediment microcosms with brominated compounds ...................................................................................................................................... 95 4.3 CHEMICAL PROPERTIES OF THE HALOGENATED ELECTRON ACCEPTOR INFLUENCE REDUCTIVE DEHALOGENATION ............... 96 4.3.1 The influence of secondary substituents onto reductive dehalogenation ....................................... 97 4.3.2 Chemical properties of heterocycles influence reductive dehalogenation ...................................... 99 4.3.3 The role of the halogen type in reductive dehalogenation ........................................................... 100 4.3.4 Chemical properties governing dehalogenation pathways of strain CBDB1 suggest halogen–
cobalamin interaction during reductive dehalogenation .............................................................. 101 4.3.5 Implication for other reductively dehalogenating microorganisms .............................................. 103 4.3.6 Potential applications and usage of the findings for fate prediction of halogenated compounds in the environment ............................................................................................................................ 104 4.4 NON‐HALOGENATED ELECTRON ACCEPTORS OF DEHALOCOCCOIDIA IN MARINE SEDIMENTS ....................................... 105 4.5 IMPLICATION FOR DEHALOCOCCOIDIA FROM MARINE SEDIMENTS ......................................................................... 108 5 CONCLUSION ............................................................................................................................... 110 6 OUTLOOK..................................................................................................................................... 111 7 SUPPLEMENT ............................................................................................................................... 113 LITERATURE .............................................................................................................................................. 144 xi
Introduction
1
Introduction
1.1
The class Dehalococcoidia
1.1.1
Isolated and cultivated strains of the class Dehalococcoidia
The bacterial class Dehalococcoidia (previously referred to as the Dehalococcoides-related
Chloroflexi (1)) belongs together with the classes Anaerolineae, Caldilineae, Ktedonobacteria,
Thermomicrobia, and Chloroflexia, to the deeply branching phylum Chloroflexi (2-5). The species
Dehalococcoides mccartyi, Dehalogenimonas lykanthroporepellens, Dehalogenimonas alkenigignens,
“Dehalobium chlorocoercia” and many sequences of so far uncultivated microorganisms from marine
and terrestrial subsurface sites constitute the Dehalococcoidia class (1, 6). More than seven strains of
Dehalococcoides mccartyi have been isolated, including strain 195, CBDB1, VS, BAV1, FL2, MB
and GT (7-16). For each of the species Dehalogenimonas lykanthroporepellens and Dehalogenimonas
alkenigignens two isolates are cultivated (17, 18). Strain DF-1 is the only cultivated representative of
“Dehalobium chlorocoercia” (19). Most cultivated Dehalococcoides and Dehalogenimonas strains
have been isolated from terrestrial freshwater sites, among them river sediments, aquifers or digester
sludge (7-13, 17, 18, 20, 21) strain MB and “Dehalobium chlorocoercia” DF-1 were isolated from
estuarine sediments (14, 22).
The cultivation of these so far isolated Dehalococcoidia is carried out under strictly anoxic
conditions in reduced synthetic mineral salts medium, amended with vitamins, except for
“Dehalobium chlorocoercia” requiring the addition of a Desulfovibrio strain cell extract (19). Acetate,
CO2 or bicarbonate serve as carbon source and hydrogen or formate as the sole electron donor.
Halogenated aromatic or aliphatic compounds are used as sole terminal electron acceptors allowing for
energy conservation only via respiration with halogenated compounds. This process is termed
organohalide respiration. During organohalide respiration, terminal respiratory enzymes called
reductive dehalogenases transfer electrons onto the halogenated organic compound, thereby cleaving
the carbon-halogen bond (for more details on the biochemistry of these enzymes see below). Many of
the halogenated compounds used for organohalide respiration by Dehalococcoidia are highly toxic to
humans and wild life and persistent in the environment. Reductive dehalogenation often renders the
compound less toxic and/or more accessible for further degradation steps. Because all so far isolated
and cultivated Dehalococcoidia are able to transform a variety of highly toxic halogenated pollutants
and depend on organohalide respiration for energy conservation, cultivated strains of the class
Dehalococcoidia are considered as key organisms for bioremediation by bioaugmentation or
biostimulation of organohalogen contaminated sites (23). Additionally, for many halogenated
compounds no other organisms besides members of the class Dehalococcoidia are known to catalyse
their transformation. However, handling and cultivation of Dehalococcoidia strains is difficult and
1
Introduction
time consuming. Growth of Dehalococcoidia strains in culture is slow with doubling times of 0.8 to
4.1 days, oxygen is not tolerated and the electron acceptors required for cultivation are often highly
toxic. Despite this, strains of the class Dehalococcoidia seem to be robust towards starvation, as cells
were observed to recover their dehalogenation activity after months of no activity at 4°C (6).
Dehalogenation activity was observed at 15°C to 35° C, but highest activity was observed at about
30°C (6).
The morphology of cultivated Dehalococcoidia cells was revealed by transmission and scanning
electron microscopy which showed disc-shaped cells less than 1 µm wide and 0.1 to 0.2 µm thick,
with concave indentations at each side. Cultivated Dehalococcoidia cells exhibit a resistance towards
antibiotics which interfere with the synthesis of a cell wall such as ampicillin and vancomycin,
indicating they lack peptidoglycan. Cells were shown to be covered by a protein layer, resembling the
S-layer of Archaea (7, 8, 10).
On
the
16S
rRNA
gene
level,
Dehalococcoides
mccartyi
and
Dehalogenimonas
lykanthroporepellens strains share ~90% sequence identity and ~87.5% with “Dehalobium
chlorocoercia” (6). Dehalococcoides mccartyi strains share more than 98% sequence identity among
each other (6, 12). Genome sequencing and annotation revealed that Dehalococcoides mccartyi strains
contain chromosomes of 1.34 to 1.47 Mb encoding around 1500 genes for a streamlined metabolism
(24-26) while the genome of Dehalogenimonas lykanthroporepellens BL-DC-9 is slightly bigger with
1.69 Mb (27). Genes of Dehalococcoides mccartyi are highly conserved between different strains
except for a high plasticity region around the origin of replication, which contains most of the
reductive dehalogenase homologous genes (rdh) (25, 26, 28). Between 10 to 36 rdh genes on a single
genome have been identified (24, 26, 28-30), which are located in an operon coding for rdh subunits.
The high number of reductive dehalogenase homologues in the genome of cultivated Dehalococcoidia
strains compared to the size of the genome, reflects the specialisation of cultivated Dehalococcoidia
strains on reductive dehalogenation.
1.1.2
Dehalococcoidia in the marine subsurface
Marine sediments harbor an immensely large and diverse number of microbes. Subsurface drilling
programs conducted allowed the analysis of sediment cores and thereby the detection of viable cells or
membrane lipids as far as 1.9 km below the seafloor (31, 32). Recent estimations predicted between
2.9 x 1029 to 5.39 x 1029 cells in in the marine subsurface, corresponding to 0.18–3.6% of the total
global biomass (33, 34). Cells were suggested to be inactive or adapted for extraordinarily low
metabolic activity in deeper organic depleted sediment layers (35). However, several studies found
that cells are active and able to survive on low energy fluxes (36-39). Therefore, this vast ecosystem is
expected to play a crucial role in global biogeochemical element cycling, especially over geological
time scales.
2
Introduction
Insights into the bacterial composition of marine sediments were obtained by 16S rRNA gene
sequence and metagenomic analyses. Sequences affiliated with the phylum Chloroflexi are one of the
most
abundant
bacterial
groups
in
marine
sediments
together
with
sequences
of
Gammaproteobacteria, Planctomycetes and the candidate phylum JS1, with Chloroflexi representing
up to 15–20% of the bacterial communities (34, 40). Furthermore, Chloroflexi have been shown to be
ubiquitously present in marine sediments from different sites and depths, ranging from shallow
organic-rich to deep and organic-depleted sediment layers (41-45). Sequences affiliating with the class
Dehalococcoidia seem to be one of the most widespread and dominant groups within the marine
Chloroflexi, implicating a significant contribution of Dehalococcoidia to biogeochemical processes
(46-51). Although Chloroflexi have been enriched from seafloor basalts, estuarine and tidal flat
sediments (52-57), and two Dehalococcoidia isolates have been obtained from estuarine sites (14, 22),
Dehalococcoidia from marine pristine sediments have not been cultivated in a pure culture
successfully so far, narrowing the available information regarding their metabolic properties.
The closest cultivated relatives of marine subsurface Dehalococcoidia sequences are notably
Dehalococcoides mccartyi, Dehalogenimonas spp. and “Dehalobium chlorocoercia”, with up to 89%
of sequence similarity (40) (Figure 1-1). Indeed, many characteristics of the cultivated
Dehalococcoidia spp. could be advantageous in deeper marine sediment environments. This includes
their slow growth, specific metabolic pathways adapted for environments with scarce resources, a
large surface to volume ratio, and an even for bacteria of extremely small cell size of 1 µm, a small
streamlined genome, the absence of oxygen protection enzymes, and the usage of hydrogen as electron
donor, and acetate as a carbon source. Hydrogen and acetate are both available from fermenting
organisms in shallow sediments and even in deep sediments, as a result of thermal
activation/maturation and aromatisation of buried organic matter (58, 59). However, little is not known
to which extend reductive dehalogenation may explain the widespread distribution of Dehalococcoidia
in pristine sediments.
Sequences of reductive dehalogenase homologous (rdh) genes – the key enzymes for
organohalide respiration by cultivated Dehalococcoidia – were detected in marine sediments from the
southeast Pacific off Peru, the Eastern Equatorial Pacific, the Juan de Fuca Ridge flank off Oregon,
and the northwest Pacific off Japan in sediments down to 358 meters below the seafloor after whole
genome amplification of DNA from sediment samples (60). In total 32 reductive dehalogenase
homologues were identified, of which many affiliated with reductive dehalogenase sequences from
Dehalococcoides mccartyi strains 195 and CBDB1 (60, 61). Moreover, experiments with enrichment
cultures from tidal flat, estuarine sediments or contaminated lagoon sediments connected
organohalide-respiring activity with the detection of 16S rRNA genes phylogenetically related to
Dehalococcoidia in enrichment cultures (52-56).
3
Introduction
Figure 1-1: Rooted 16S rRNA gene-based phylogenetic tree of representatives of the phylum Chloroflexi. Green
branches mark the Dehalococcoides-related Chloroflexi cluster. Dehalococcoides mccartyi strain CBDB1 used
in the current work as model organism for organohalide-respiring Dehalococcoidia is highlighted in ( ). Single
amplified Dehalococcoidia genome C11 (SAG-C11), was used in the current work as model organism for
Dehalococcoidia with an organohalide respiration-independent mode of living is highlighted in ( ). The tree was
constructed using the Neighbour joining algorithm, evolutionary distances were calculated using the p-distance
method in Mega 6.0. Numbers show bootstrap values above 0.5 after 1000 replications.
Diverse Dehalococcoidia subgroups have been identified in marine sediments. All isolated
organohalide-respiring strains of the class Dehalococcoidia affiliated with one of the subgroups
termed ‘Ord-DEH’ (47). Indications of metabolic properties of Dehalococcoidia from other subgroups
have been gained from single-cell and metagenome analyses (46, 48, 62). Annotations of
Dehalococcoidia single-cell genome DEH-J10 (subgroup ‘GIF9-B’) (46), Dsc-1 and DscP2 (48), as
well as metagenomics pan-genomes RGB-2 (subgroup GIF-9) and RGB-1351 (tentatively assigned to
the GIF3 or vadinBA26 subgroup) (62) gave more detailed information about potential metabolic
properties of different Dehalococcoidia subgroups. In the genomes of DEH-J10, DscP2, RGB-2 and
RGB-1351, genes coding for enzymes of the Wood-Ljungdahl pathway were detected. For DEH-J10,
4
Introduction
RGB-2 and RGB-1351, ATP generation during the formation of acetate from acetyl-CoA, and beta
oxidation of fatty acids was furthermore predicted. In Dehalococcoidia single amplified genome
DEH-J10, genes which confer the ability to oxidize substituted aromatics and to use dimethyl
sulfoxide (DMSO) or trimethylamine N-oxide as terminal electron acceptor for respiration via a
dimethyl sulfoxide reductase were found (46). No indications for organohalide-depending modes of
living were detected in any of the genomes. In RBG-2 and DscP2, genes coding for an aerobic
haloacid dehalogenases were detected which catalyzes dehalogenation of chlorinated and brominated
substrates (63, 64). Haloacid dehalogenases however, are non-homologous to reductive dehalogenases
and are not used as terminal reductases for respiration. In DscP2 a gene encoding a protein with
homology over an Fe-S cluster domain of known respiratory RdhA was detected, but a TAT leader
sequence and a typically adjacent rdhB gene coding for a membrane anchor subunit was missing (48).
1.2
Organohalide respiration
Halogenated compounds are introduced to the environment from anthropogenic sources as
pesticides, flame retardants, additives to polymers and pharmaceuticals and during the production of
dyes, agrochemicals and solvents (65-67). To date many halogenated compounds have been banned
owing to their high toxicity and negative effects on humans and the environment. However,
halogenated compounds often persist in the environment long time after their prohibition as a result of
their recalcitrant properties and their potential to bioaccumulate. Accordingly, they can be widely
detected not only in sediments and water, but also in tissues and milk of animals and humans (68-73).
Many naturally occurring halogenated compounds are also known to exist (described below).
Transformation of halogenated compounds highly depends on the environment. In oxic
environments, transformation of halogenated alkenes, short-chain alkanes and some aromatic
compounds was reported involving mono- and dioxygenases and molecular oxygen in metabolic and
co-metabolic processes (74-76). A preference for halogenated organic compounds with a low number
of halogen substituents was observed for metabolic aerobic dehalogenation (77). Recently, the
reductive dehalogenation of halogenated compounds in aerobic environments was also described (78).
In anoxic environments, several bacterial groups that are able to catalyse reductive dehalogenation
reactions have been identified (7, 8, 10, 79-82). Members of the Delta- and Epsilonproteobacteria are
facultative organohalide-respiring bacteria and the organohalide-respiring members of these phyla can
also grow by fermentation. The phylum Firmicutes comprises both obligate and facultative
organohalide-respiring organisms. All isolated and cultivated strains of the class Dehalococcoidia
from the phylum Chloroflexi are obligate organohalide-respiring organisms. Values for changes in
Gibbs free energy during reductive dehalogenation of various halogenated aromatic and aliphatic
compounds suggested a similar energy yield for halogenated alkyl and aryl compounds as for the
nitrate/nitrite couple when used as terminal electron acceptor for respiration in anoxic environments.
5
Introduction
In contrary to aerobic dehalogenation, a preferential dehalogenation of higher halogenated compounds
compared to less halogenated compounds as terminal electron acceptor in anoxic environments was
predicted (83). In rather rare cases, halogenated compounds were reported to be transformed by other
processes than reductive dehalogenation under anoxic conditions (84, 85).
Co-metabolic dehalogenation, in which other energy sources or electron acceptors are required
and only low dehalogenation rates are observed, has been reported predominantly for aliphatic
halogenated compounds in anoxic environments (86). The very toxic and common contaminant vinyl
chloride is dehalogenated co-metabolically by Dehalococcoides mccartyi strain 195 but can be used
for growth in a metabolic transformation by strain BAV1 (8, 10). Co-metabolic reductive
dehalogenation was also observed for sulphate reducers (87), methanogenic Archaea (88) or acetogens
(89).
For the metabolic transformation of halogenated compounds by Dehalococcoidia hydrogen serves
as electron donor, except for ‘Dehalobium chlorocoercia’ using formate as electron donor (6). The
electron transport between hydrogenase and reductive dehalogenase is coupled to the generation of
ATP and bacterial growth. For Dehalococcoidia the electron shuttle between hydrogenase and
reductive dehalogenase has not been identified so far. No cytochrome-containing proteins or
cytochrome biosynthesis genes have been identified in Dehalococcoides mccartyi strains (24-26).
Quinones serve as electron shuttle between hydrogenase and reductive dehalogenase in organohaliderespiring Desulfitobacterium hafniense (90), however, no evidence was found that quinones are
involved in organohalide respiration in Dehalococcoides mccartyi strain CBDB1 (91).
Reductive dehalogenases are the key enzymes in organohalide respiration. A main difference
between the genomes of Dehalococcoides mccartyi strains is the number and type of reductive
dehalogenase operons. A range of 10 to 36 genes encoding rdh genes on a single genome have been
identified (24, 26, 28-30). Generally, rdh genes are located in islands around the origin of replication
and are located in an operon structure coding for various Rdh subunits. RdhA is a 50–65 kDa protein
that functions as the catalytically active enzyme. The N-terminus of most encoded RdhA harbour twin
arginine transport (TAT) signal peptide motifs for the translocation of folded proteins to or across the
inner cell membrane. At the C-terminus of the RdhA two conserved motifs can be found, with four
cysteine residues that can form iron sulphur clusters (92). Adjacent to the rdhA, the rdhB is localized,
coding for a small hydrophobic protein of about 10 kDa with 2-3 predicted transmembrane helices. It
is predicted to function as a membrane anchor protein for the RdhA subunit (29). This type of
organization was not only observed in Dehalococcoides mccartyi but also in Dehalogenimonas,
Dehalobacter, Desulfitobacterium and Sulfurospirillum (9, 11, 93-98). However, exceptions were
described in which rdhA does not exhibit a TAT leader sequence or in which an adjacent rdhB gene
was missing (20). Typically rdh genes are associated with a MarR-type or two component-type
transcriptional regulator in Dehalococcoides species, however, there are exceptions in which the rdh
6
Introduction
genes are not directly associated with a regulator as for instance in Dehalogenimonas
lykanthroporepellens type strain BL-DC-9 (27).
The induction and substrate specificity of reductive dehalogenases towards different halogenated
compounds is described for only few enzymes compared to their abundance and diversity encoded by
Dehalococcoidia genomes. Low biomass yields and the inactivation of reductive dehalogenase
enzymes by oxygen rendered purification of reductive dehalogenases a complicated step. The
heterologous expression of active reductive dehalogenase was not possible until very recently (99,
100). Transcription studies were used to investigate reductive dehalogenase induction under specific
cultivation conditions (97, 101, 102). In Dehalococcoides mccartyi strain CBDB1 for instance the
transcription of cbdbA1453 and cbdbA187 was enhanced in the presence of 1,2,3-trichlorobenzene
whereas the transcription of cbdbA1624 was enhanced by 1,2,4-trichlorobenzene (103). The reductive
dehalogenase CbrA (cbdbA84) was isolated by native gel electrophoresis and characterized after
growth
of
strain
CBDB1
with
1,2,3-trichlorobenzene
was
shown
to
be
active
with
1,2,3-trichlorobenzene in methyl viologen-based activity assays (104). The tetrachloroethene reductive
dehalogenase PceA and the trichloroethene reductive dehalogenase TceA were identified in
Dehalococcoides mccartyi strain 195 which catalyse the reaction of tetrachloroethene to
trichloroethene and trichloroethene to ethene, respectively (105). In strain VS the reductive
dehalogenase VcrA was shown to be active with vinyl chloride (9) and the MbrA reductive
dehalogenase from strain MB transformed tetrachloroethene and trichloroethene to transdichloroethene (101). Beyond this, proteome-based approaches were used to investigate the expression
of reductive dehalogenases in presence of specific electron acceptors and to assign putative functions
to the enzymes (106, 107). Similar to transcriptional studies, it is particularly difficult to elucidate the
function of a single reductive dehalogenase, since multiple reductive dehalogenase are typically
expressed even in the presence of a single substrate (103, 108).
The active cofactor of reductive dehalogenases was shown to be a corrinoid in purified reductive
dehalogenases (93, 94, 105, 109, 110). Inhibition studies with whole cells and alkyl-iodides allowed a
light reversible inhibition of dehalogenation activity, indicating the involvement of a corrinoid in the
active site of the dehalogenases (105, 111). The only so far known exception is the 3-chlorobenzoate
reductive dehalogenase of Desulfomonile tiedjei, which contains iron in the catalytic centre (112).
Different types of corrinoid cofactors have been detected in organohalide-respiring organisms. For
Dehalococcoides mccartyi strain 195, for instance, indications for a standard cobalamin cofactor were
found (113), while a tetrachloroethene reductive dehalogenase isolated from Sulfurospirillum
multivorans contained a norpseudovitamin B12 (114). The standard redox potential of the
cob(III)alamin/cob(II)alamin couple is +204 mV and of the cob(II)alamin/cob(I)alamin -606 mV. The
low redox potential of the Co(I)/Co(II) couple, together with the observation that cyanocobalamin can
catalyse dechlorination reactions in vitro and the inhibition of reductive dehalogenation by alkyl
iodides lead to the assumption that the corrinoid reacts in the Co(I) state with the organohalogen.
7
Introduction
Corrinoids are strong nucleophils and reaction mechanisms involving nucleophilic substitutions
(115) and mechanisms including radical intermediates have been proposed (116). So far considered
reaction mechanisms assume an attack of the electron onto the partially positively charged carbon
atom substituted by the halogen that will be removed by reductive dehalogenation. While inhibition
studies with alkyl iodides suggest the formation of an organocobalt adduct, other experimental
evidence suggests an electron transfer by the corrinoid onto the organohalogen without the formation
of an organocobalt intermediate leading to a radical anion which is subsequently reduced by a second
electron possibly from the oxidation of Co(II) to Co(III) (90). Very recently electron paramagnetic
resonance spectroscopy and simulation of the electron acceptor-reductive dehalogenase interaction
suggested a halogencobalamin interaction in the active site of a soluble oxygen tolerant reductive
dehalogenase (117).
1.3
Electron acceptors for Dehalococcoidia
1.3.1
Electron acceptors of cultivated Dehalococcoidia
Whereas several strains of the class Dehalococcoidia reductively dehalogenate aliphatic
compounds such as chlorinated ethenes (8, 10), other strains of the class Dehalococcoidia are
specialized on reductive dehalogenation of chlorinated aromatics (7). Dehalococcoidia mccartyi strain
CBDB1 for instance requires a conjugated pi-system with halogens as substituents that are replaced
during reductive dehalogenation by hydrogen (7, 118-121). In contrast to Dehalococcoides mccartyi
strains, Dehalogenimonas strains dehalogenate a variety of vicinally polychlorinated alkanes in a
dihaloelimination reaction. During reductive dehalogenation via hydrogenolysis, only one halogen is
removed, while during dihaloelimination two halogens are removed in one step, thereby establishing a
pi-bond between the dehalogenated carbon atoms (18, 20, 27 ).
Reductive dehalogenation of halogenated electron acceptors follows specific pathways when
catalysed by different strains of the class Dehalococcoidia. The reasons for the specificity of the
pathways are not fully understood. To describe the preferential dehalogenation pathways, halogens
have been classified as doubly flanked, singly flanked or isolated (122). Dehalococcoides mccartyi
strain CBDB1, for instance, preferentially removes halogens that are doubly flanked in chlorinated
benzenes and biphenyls (7, 118). Chlorine atoms with only one neighbouring halogen atom were
dehalogenated with a lower rate or not removed at all. Isolated chlorine atoms were not dehalogenated
(123, 124). For Dehalococcoides mccartyi strain CBDB1 a similar effect on reductive dehalogenation
was
described
for
a
hydroxy
substituent
compared
to
a
halogen
substituent
(119).
2,3,4-Trichlorophenol was dehalogenated by strain CBDB1 to 3,4- and 2,4-dichlorophenol (119).
However, 3,4-dichlorophenol was dehalogenated only in the presence of a higher chlorinated phenol,
while 1,2,4-trichlorobenzene was dehalogenated to 1,3- and 1,4-dichlorobenzene, showing that the
effect of the hydroxy group on reductive dehalogenation was weaker than the one of a flanking
8
Introduction
halogen (7, 119). Similarly, a positive effect of oxygen heteroatoms on reductive dehalogenation was
observed for chlorinated dioxins (121).
Substituent effects might have implications for the natural electron acceptor range available for
Dehalococcoidia. Humic acids and fulvic acids detected in sediments, natural water bodies and soils
using in-situ X-ray spectroscopy, enclose aromatic and aliphatic organochlorines with phenolic and
aliphatic groups (125). The concentration of organochlorine compounds in humified plant material
was estimated to be between 0.5 and 2 mM kg-1, and aromatic organochlorine compounds have been
suggested to be the more abundant ones compared to aliphatic organochlorines (125). Different
functional groups of natural halogenated compounds might influence whether a halogenated molecule
can be used as electron acceptor by organohalide-respiring Dehalococcoidia. Indeed, the number of
Chloroflexi 16S rRNA genes copies were shown previously to positively correlate with organochlorine
per total organic carbon and to increase in numbers parallel to chloride release in cultures incubated
with organochlorines (126).
1.3.2
Natural organohalogens as potential electron acceptors in marine sediments
Organohalogens occur naturally in the environment as a result of abiotic and enzymatic reactions
(127, 128). In marine environments, brominated compounds dominate over chlorinated compounds
while in terrestrial environments chlorinated compounds seem to be produced in larger quantities
(129). Chlorinated compounds such as PCE and TCE are produced as secondary metabolites by
marine algae. Subsurface volcanic emissions, pyrogenic activity, haloperoxidase of fungi, reaction
during plant weathering and abiotic alkylation of halides during Fe(III) oxidation, produce chlorinated
and brominated compounds, which can be detected in marine waters (125, 127, 130, 131). For
brominated compounds, up to 2,000 natural molecules have been identified, mostly from marine origin
(131). Their production has been shown for macroalgae, phytoplankton, hemichordates, sponges, and
molluscs which use them most likely as chemical defence mechanisms (128, 132-136). Marine
organisms seem to produce in particular a range of low molecular weight brominated aromatic and
aliphatic compounds including bromophenols, brominated furanones, tribromoanisole, bromopyrroles,
bromoalkanes, bromoketones and bromophycolides, and polybrominated diphenyl ether analogues
such as 4,6-dibromo-2-(2,4-dibromo)phenoxyanisole (128, 132, 133, 136-138) (Figure 1-2).
The identification of the specific molecular structure of organobromines in marine sediments is
difficult owing to the high diversity and low concentration of each individual compound in marine
sediments. Synchrotron‐based X‐ray spectroscopic and spectromicroscopic techniques were used
however, to investigate organobromine distribution and speciation in coastal and deep marine
sediments (139). It was shown that concentrations of brominated organic compounds were high in
shallow sediments but decreased with increasing sediment depth correlating with the concentration of
non-halogenated organic compounds, while inorganic bromine concentrations increased with depth.
9
Introduction
This could be due to abiotic reactions but also microbial catalysed reductive dehalogenation with a
release of inorganic bromine into the pore water (140, 141).
Figure 1-2: Examples of brominated compounds isolated from mussels, algae and sponges (128, 132, 133, 136138).
Dehalogenation of brominated compounds was observed in marine and estuarine sediments or
marine organobromine producing sponges (60, 140, 141), but so far debromination of aromatic
compounds was not specifically connected to growth of Dehalococcoidia from oceanic marine sites.
Debromination of brominated aromatics by members of the class Dehalococcoidia class was
described for a co-culture containing Dehalococcoides and Desulfovibrio species debrominating
diphenyl ethers (15). However, Desulfovibrio species were described to dehalogenate bromo- and
iodophenols (141, 142). Therefore, the participation of Desulfovibrio spp. in the debromination
reaction could not be excluded. D. mccartyi strain BAV1 and 195 were shown to dehalogenate
brominated biphenyl. However, debromination was incomplete and occurred only in presence of a
chlorinated aliphatic compound (143). The dehalogenation of brominated aliphatic compounds by
Dehalococcoidia strains was described for Dehalococcoidia strain BAV1 and vinyl bromide (10) and
for alkyl bromines in activity assay-based studies (95).
1.3.3
Non-halogenated electron acceptors for Dehalococcoidia in marine sediments
16S rRNA gene sequences affiliating with the class Dehalococcoidia were detected in different
sediment environments and the relative abundance of the different Dehalococcoidia subgroups were
described to shift with geochemical gradients e.g. with the sulphate-methane transition zone (47).
Moreover, reductive dehalogenase genes were absent in several Dehalococcoidia single-cells or
metagenome-derived pan-genomes (46, 144). Therefore, other electron acceptors besides halogenated
compounds may contribute to the abundance and widespread distribution of Dehalococcoidia in
marine sediments.
In most marine sediments, with moderate to high organic inputs, oxygen is depleted rapidly by
aerobic respiration within the first few millimetres or centimetres below the seafloor. With increasing
sediment depth, electron acceptors for anaerobic respiration are used in a succession of decreasing
energy yields. Typically, nitrate, manganese(IV), iron(III), sulphate, and CO2 are used, while other
less-studied electron acceptors such as elemental sulphur, other sulphur species (e.g. thiosulphate) and
10
Introduction
even rare elements such as arsenate(V) or organic molecules such as dimethylsulphoxide (DMSO) or
trimethylamine-N-oxide (TMAO), can be reduced in respiratory processes by prokaryotes in marine
sediments.
Sulphate reduction is considered as one of the most important processes for anaerobic
mineralization of organic matter in marine sediments (145). In the assimilatory sulphate reduction
pathway, sulphate is reduced to sulphide, then incorporated into cysteine and subsequently processed
to a range of other sulphur-containing biomolecules. Sulphate assimilation is a widespread process
found in all three domains of life. In the dissimilatory pathway of sulphate reduction, sulphate is used
as a terminal electron acceptor for respiration. Sulphate respiration was identified in four bacterial
phyla, namely Proteobacteria, Firmicutes, Nitrospirae, Thermodesulfobacteria, and the two archaeal
phyla Euryarchaeota and Crenarchaeota (146-150).
Because sulphate reducers are polyphyletic, 16S rRNA gene-targeting primers, which cover the
16S rRNA genes of all sulphate reducers are not available. Alternatively, functional genes of the
adenylyl-sulphate-reductase adenosine-phosphosulphate reductase (APR reductase) or dissimilatory
sulphite reductases (drsAB), both involved in sulphate reduction, have been used extensively as a
functional gene marker to investigate the distribution, diversity, and evolution of sulphate reducers
(151-158). APR and Dsr are both key enzymes in the anaerobic sulphate respiration and are present in
all known sulphate reducing organisms. Dissimilatory sulphite reductase genes were also detected in
members of the Proteobacteria and Firmicutes which are unable to respire sulphate. They have been
reported to use sulphite as terminal electron acceptor (159), disproportionate inorganic sulphur
compounds (160) or metabolize organosulphonates to internally produce sulphite for respiration (161).
For few dsrAB-containing Firmicutes genera, the function of dsrAB is unknown (162).
Comparative amino acid sequence analysis of the dissimilatory sulphite reductase genes and
phylogenetic congruence of the DsrAB tree with the 16S rRNA tree, revealed a mostly vertical
inheritance of dissimilatory sulphite reductases (151). However, for some groups 16S rRNA and
dsrAB phylogenies were not congruent. Archaeoglobus, several Desulfotomaculum species and the
genus Thermodesulfobacterium likely obtained their dsrAB genes via a lateral gene transfer (157).
The usage of dissimilatory sulphite reductase genes as a functional marker has revealed high dsr
sequence diversity in different environments. Many of the amplified sequences belong to uncultured
sulphate-reducing microorganisms and have been shown to group into at least thirteen stable
family-level lineages, which do not contain any cultivated representatives (150). Therefore, the
potential for sulphite/sulphate reduction is expected to be more widespread in different bacterial taxa
than so far recognized (150, 152, 158). For instance, sediment sites with high sulphate reduction
activity but few dsrA gene copy numbers or 16S rRNA genes of sulphate reducing prokaryotes were
described (49, 163). It was suggested that either sulphate reducing bacteria populations are small but
active, or that sulphate reduction is performed by yet unknown sulphate reducing organisms with
divergent genes, so far not detected by common primer and PCR methods (40, 154, 163).
11
Introduction
1.4
Goal of this work
This work aimed at contributing to the understanding of the class Dehalococcoidia with respect to
their widespread occurrence in marine sediments and the ability of several cultivated members of this
class to transform halogenated compounds. Thereby the work was divided into two main parts which
focused on one hand on the establishment of methods to investigate the potential of marine
Dehalococcoidia to grow by organohalide respiration using D. mccartyi model organism CBDB1 and
on the other hand on the potential of Dehalococcoidia to gain energy via an organohalide
respiration-independent mode of living. For this purpose, electron acceptors were investigated that
could support respiration of Dehalococcoidia in pristine marine and terrestrial sediments. This was
achieved by studying reductive dehalogenation and growth of Dehalococcoidia model organism
D. mccartyi strain CBDB1 with halogenated electron acceptors to elucidate fundamental chemical
parameter which influence reductive dehalogenation and might therefore serve to identify potential
natural electron acceptors of Dehalococcoidia in non-contaminated environments and to predict the
fate of halogenated compounds from natural but also anthropogenic sources in anoxic environments.
Additionally, a microtiter plate format activity assay was established with D. mccartyi strain CBDB1
to provide a fast an easy screening tool to identify new electron acceptors of organohalide-respiring
Dehalococcoidia and marine dehalogenating bacterial strains and to determine transformation rates of
electron acceptors. Furthermore, strain CBDB1 was studied for activity and enzymatic expression with
brominated benzenes as model compound for commonly occurring brominated compounds in marine
environments. Chemical descriptors of halogenated compounds were evaluated to gain insight into
chemical parameters, governing reductive dehalogenation. The amplification of functional respiratory
genes from marine sediments was used additionally to study the organohalogen-independent
respiration of Dehalococcoidia subgroups with non-halogenated electron acceptors in marine
sediments. Together the research gives insight into modes of anaerobic respiration catalysed by
members of the class Dehalococcoidia.
12
Material and Methods
2
Material and Methods
2.1
Chemicals and gases
Chemicals were purchased from Carl ROTH (Karlsruhe, Germany), Sigma-Aldrich (Steinheim,
Germany) and Merck (Darmstadt, Germany). Solutions and container were sterilized by autoclaving at
121°C for 20 min or by filtering through a 0.2 µm pore size Minisart filter (Sartorius, Göttingen,
Germany). Container and solutions for molecular work were additionally treated by exposure to UV
light for 30 min. All experiments requiring anoxic conditions were carried out within an anoxic
chamber (Coy-Labs, Grass Lake, Michigan, USA) filled with nitrogen of 99.999% v/v purity and 2–
4% hydrogen. TE-buffer was prepared with Millipore water and 10 mM Tris adjusted to pH 8.0 with
HCl and 1 mM EDTA. For the 0.9% NaCl solution 9 g of NaCl were added to 1000 ml of Millipore
water.
Table 2-1: Gases used for cultivation
Compound
Helium 5.0
Nitrogen 4.0
Hydrogen 5.0
Biogon ® C 20 (80% N2 20% CO2)
Aligal (20 ± 2 Vol.% CO2 in N2)
Forming gas 80/20 (20 ± 2% H2 in N2)
2.2
2.2.1
Company
Air Liquide Deutschland GmbH, Düsseldorf,
Germany. Linde AG, Munich, Germany
Bacterial cultures and inocula
Cultures
Table 2-2: Different cultures and strains used in the current study
Strain
D. mccartyi strain CBDB1
Sediment microcosms from
the coast off Chile and Peru
Genotype/Phenotype
Wild type
Uncharacterized wild type
NEB 5-alpha competent
E. coli K12 strain (C2987)
fhuA2 Δ(argF-lacZ)U169 phoA
glnV44 Φ80 Δ(lacZ)M15
gyrA96 recA1 relA1 endA1 thi1 hsdR17
13
Reference/company
(7)
Camelia Algora, Helmholtz
Centre for Environmental
Research, personal contact
New England Biolabs GmbH,
Frankfurt am Main, Germany
Material and Methods
2.2.2
Sediment samples
Table 2-3: Origin of sediment samples
Sample
source
Cruise
Collection
date
Site
Aarhus,
Denmark
Baffin Bay,
Greenland
Wadden
Sea,
Hedwigenk
oog,
Germany
Black Sea
-
2011
Mimosa
ARK-XXV/3
2010
371
-
2011
M72/1
2007
214SL
Peru
ODP Leg
201
SO-156/3
2002
1227
2001
GeoB
7155-4
Chilean
continental
margin
Site coordinates
(latitude,
longitude)
56°09.60'N,
10°28.10'E
75º58.24'N,
70°34.86'W
54°11'25.6"N
8°48'51.0"E
Water
depth (m)
16.3
Deepest
sample
(mbsf)
3.0
598
4.05
0–2
0.3
4°24.10'N,
32°51.27'E
8°59.5'S,
79°57.40'W
34°35.00'S,
72°53.11'W
1680
3.60
427
121.20
2744
6.70
Sediments of the Aarhus Bay were sampled with a gravity corer (3 m) at water depth of 16.3 m
(see also Table 2-4). After retrieving, the core was cut into layers and immediately pore water was
withdrawn for methane and sulphate measurements. Additionally, 10 ml of sediments were withdrawn
with 10 ml plastic syringes (Braun, Melsungen, Germany) in triplicates, sealed in plastic bags and
frozen at -20°C for molecular analyses in the laboratory. The rest of the sediments were stored in
gas-tight sealed container or in gas-tight serum Schott bottles. Sediments for cultivation or proteomics
were stored at 4 °C.
Figure 2-1: Withdrawal of a sediment core from the bay of Aarhus using a 3 m gravity corer.
14
Material and Methods
Table 2-4: Sediment core samples
„Name“
1
A
2
B
3
C
4
D
5
E
6
F
7
G
8
H
9
I
1
Depth [cm]
0–30
30–36
36–66
66–72
72–102
102–108
108–135
135–141
141–168
168–174
174–201
201–208
208–235
235–241
241–268
268–274
274–301
301–308
Purpose
Cultivation/Proteomics
Molbiol1/Sulphate/Methane
Cultivation/Proteomics
Molbiol/Sulphate/Methane
Cultivation/Proteomics
Molbiol/Sulphate/Methane
Cultivation/Proteomics
Molbiol/Sulphate/Methane
Cultivation/Proteomics
Molbiol/Sulphate/Methane
Cultivation/Proteomics
Molbiol/Sulphate/Methane
Cultivation/Proteomics
Molbiol/Sulphate/Methane
Cultivation/Proteomics
Molbiol/Sulphate/Methane
Cultivation/Proteomics
Molbiol/Sulphate/Methane
Storage
4°C and -20°C upper 10 cm
-20°C
4°C
-20°C
4°C
-20°C
4°C
-20°C
4°C
-20°C
4°C
-20°C
4°C
-20°C
4°C
-20°C
4°C
-20°C
for molecular biological applications
2.3
Cultivation
2.3.1
Medium preparation for anaerobic cultivation
2.3.1.1 Preparation of the reducing agent titanium(III) citrate
Titanium(III) citrate was prepared in nitrogen atmosphere according to Zehnder and Wuhrmann
(164). For this 5.14 ml 15% titanium(III) chloride solution were added to 10 ml anoxic 1 M sodium
citrate solution. Solid sodium carbonate was used to neutralize the solution to pH 7 and anoxic water
was added to a final volume of 50 ml to obtain a final concentration of 0.1 M titanium(III)chloride and
0.2 M sodium chloride.
2.3.1.2 Preparation of 1 M sodium hydrogen carbonate buffer
Sodium hydrogen carbonate (7.06 g) was added to a 100 ml serum bottle and 84 ml of anoxic
Millipore water was added. The bottle was sealed with a rubber septum. The mixture was flushed for
several minutes with CO2 with the help of a needle and shaken several times with the lid closed. This
procedure was repeated until the carbonate was dissolved completely. The bottle was closed with an
aluminium crimp cap and autoclaved. Before usage the solution was cooled to room temperature to
avoid precipitation of carbonate minerals in the media.
15
Material and Methods
2.3.1.3 Preparation of salt and vitamin solutions
Table 2-5: solutions for anaerobic cultivation. All solutions were prepared in Milli-Q water.
Mineral salt solution
(Widdel fresh water solution)
Compound
Final conc.
g L-1
KH2PO4
10
Trace element solution SL9
Modified from (165)
Compound
Final conc.
mg L-1
Nitrilotriacetic 12800
acid (NTA)
FeCl2 x4H2O
2000
ZnCl2
70
NH4Cl
13.5
NaCl
50
MnCl2 x2H2O
80
MgCl2 x
6H2O
KCl
CaCl2 x
2H2O
20.5
H3BO3
6
26
7.5
CoCl2 x6H2O
CuCl2 x2H2O
190
2
NiCl2 x6H2O
Na2MoO4
x2H2O
NaOH ad pH
7.0
Sterilized by
autoclaving
24
36
Sterilized by
autoclaving
Vitamin 7 solution
Compound
D(+)-Biotin
Nicotinic acid
Ca-D (+)Pantothenic acid
Pyrodoxine
hydrochloride
Thiaminechloridedihydrochloride
Cyanocoalamin
4-Amino benzoic
acid
Final conc.
mg L-1
2
5
5
10
5
50
5
Filter sterilized
2.3.1.4 Preparation of cultivation medium for Dehalococcoides mccartyi strain CBDB1
The medium for cultivation of strain CBDB1 was prepared by adding Widdel mineral salt
solution, trace element solution SL9, sodium acetate solution, selenite-tungstate solution and resazurin
to Milli-Q-water (see Table 2-6). The solution was degased for 30 min in an anoxic gas exchange
chamber and purged subsequently for 30 min with nitrogen outside the anoxic glove box. In an anoxic
glove box the liquid was dispensed into serum bottles or Schott bottles. Unless stated otherwise 50 ml
bottles with 30 ml liquid volume were selected for cultivation to obtain a headspace to liquid ration of
about 2:3. After equilibration of media for 1 h in the anoxic glove box, the bottles were sealed with
Teflon-lined butyl rubber stopper, crimped with aluminium caps, and autoclaved. After the liquid was
cooled to room temperature, NaHCO3 buffer (1:100 (= 1% vol/vol)) and titanium(III) citrate (1:100 (=
1% vol/vol)) were added with a plastic syringe (Braun, Melsungen Germany) connected to a 0.2 µm
sterile filter (Minisart®, Sartorius AG, Göttingen, Germany) under a clean bench or near a Bunsen
flame. The medium was equilibrated in the flasks for at least 4 hours. Before inoculation of strain
CBDB1, vitamin-7 solution was added freshly from a sterile 100x stock solution (prepared from a
1000x-concentrate in anoxic water).
16
Material and Methods
Table 2-6: Medium components
Component
Milli-Q-water
Widdel mineral salt solution (50x conc.)
Trace element solution SL9
Sodium acetate solution 1 M
Resazurin
Selenit-tungstate solution
NaHCO3 1 M
15% Titanium(III) citrate
Vitamin 7 solution (100x conc.)
Milli-Q-water
Added amount or final concentration
800 ml
20 ml
10 ml
5 ml (5 mM)
5–10 drops (~1 µM)
1 ml (~ 0.025 µM each)
10 ml (10 mM)
10 ml (~2 mM)
10 ml
filled up to 1000 ml
2.3.1.5 Addition of halogenated compounds
The halogenated compounds were added to the medium with a 10 µl glass syringe (Hamilton,
Bonaduz, Switzerland) from an acetonic stock solution or they were added as crystals. The
concentration of electron acceptor in the medium was determined by gas or ion chromatography after
equilibration of at least one hour.
Table 2-7: Compounds tested with strain CBDB1 in cultivation
Compound
Solvent
Aromatic compounds with non-halogen substituents
2,3-; 2,4-; 2,6-dichloroaniline
acetone
2,3-; 2,5-; 2,6-dichlorobenzonitrile
2-chloro-6-fluorobenzonitrile
2,4,6-tribromophenol
acetone
2,4-; 2,6-dibromophenol
acetone
4-bromo-3,5-dimethoxybenzoic acid
4-bromo-3,5-dihydroxybenzoic acid
Heterocyclic aromatic compounds
4,5-dibromo-2-furoic acid
5-bromo-2-furoic acid
2,3-; 2,6-dichloropyridine
acetone
Concentration in
the stock solution
[mM]
Final
concentration
[µM]
1M
100 µM
added as crystals
added as crystals
1M
1M
10 µM
10 µM
added as crystals
added as crystals
added as crystals
added as crystals
1M
50 µM
Cultures were initially set-up from cultures grown on 30 µM 1,2,3-TCB. A sterile 10 ml plastic
syringe (Omnifix®, Braun, Melsungen, Germany) flushed previously with sterile, anoxic H2O, was
used for inoculation. An inoculum percentage of 10% was used for strain CBDB1 corresponding to
starting cell densities of 5 x 105 to 5 x 106 cells ml-1. The headspace was pressurized with 20% CO2 /
80% N2 (1.3 bar), and 0.1 bar hydrogen was added to obtain a final pressure of 1.4 bar. Cultivation
was performed in the dark at 30°C without shaking. All cultivations were done at least in triplicates.
Additionally, chemical controls without inoculum, negative growth controls without electron acceptor
and positive controls with 30 µM 1,2,3-TCB were set up. Cultures were monitored by cell counting
and gas or ion chromatography.
17
Material and Methods
2.3.2
Cultivation of E. coli
E. coli cells used for cloning experiments were cultivated in liquid Luria-Bertani medium (1%
trypton, 0.5% yeast extract, 1% NaCl, pH 7) or on Luria-Bertani agar plates (LB-medium, 1.5% agar).
The medium was set up with Milli-Q-water and autoclaved. After cooling to ~50°C, ampicillin (final
conc. 100 µg ml-1) was added under a clean bench. For the identification of positive clones with bluewhite screening X-Gal was added to the medium to a final concentration of 80 µg ml-1. For agar plates,
approximately 25 ml of LB-agar medium was filled into each petri dish, dried under the clean bench
and stored at 4°C for further usage.
The medium was inoculated with a single colony from an ampicillin/X-Gal-containing LB-agar
plate and incubated over night at 37°C and 250 rpm. E. coli was cultivated on LB-agar plates at 37°C
for at least twelve hours. E. coli colonies were stored on LB-agar plates at 4°C.
2.3.3
Direct microscopic counting
Cell numbers were determined by direct epifluorescence-microscopic counting after staining with
SYBR-green (Invitrogen, Carlsbad, California, USA) on agarose-coated slides. For the preparation of
the slides two grams of low-melting SeaPlaque® agarose (Biozym Scientific GmbH, Hessisch
Oldendorf, Germany) and 120 ml water were stirred while being heated carefully until the agarose was
liquefied completely. The liquefied agarose was withdrawn with a pre-warmed 10 ml glass pipette and
glass slides were covered with approximately 2 ml of agarose and dried under the clean bench (119).
The SYBR1 Green double strand DNA stain (Invitrogen, Carlsbad, California, USA) was diluted
1:100 with sterile TE-buffer (10 mM Tris, 1 mM EDTA, pH 7.2) and stored in 0.5 ml Eppendorf tubes
in 10 µl aliquots at -20°C. For counting medium was withdrawn from a culture with a 1 ml plastic
syringe (BD Plastipak™, Becton Dickinson GmbH, Heidelberg, Germany) and 20 µl were mixed with
1 µl diluted SYBR green stock solution, resulting in a final concentration of 1:2000 SYBR green in
the sample. The tube was incubated in the dark for 10 min. For counting, 18 µl were pipetted on a 20
mm x 20 mm cover slide. By this procedure the cells were immobilized in one defined focus level
between the cover slide and an agarose-covered glass slide (119). Cell numbers were quantified by
direct microscopical counting using epifluorescence of SYBR-green stained cells as described
previously (119). Micrographs were taken using a Nikon fluorescence microscope at 400x
magnification with a NikonDS-Ri1 digital camera. For calibration a micrometre scale was
photographed with the same parameters as applied for sample micrographs. For each sample fifteen
micrographs were taken from randomly chosen positions. The micrographs were taken with the NIS
imaging software (Nikon, Tokyo, Japan). Cell counting and calculation of picture size were evaluated
by using ImageJ (http:// http://imagej.nih.gov/ij/) mainly automated by ImageJ and Excel macros
(119).
18
Material and Methods
2.4
Preparation of whole cells suspensions and detachment of cells from sediments
2.4.1
Rotary evaporator
Rotary evaporation was used to concentrate cells and to remove halogenated solvents from the
cell suspension. Approximately 30 ml cell culture were transferred in an anoxic glove box into a 500
ml round bottom flask and sealed with a glass stopper. The flask was removed from the glove box and
connected to a rotary evaporator. The culture was concentrated to approximately half of the original
volume at 50 mbar. A water bath heated to 30°C was used to accelerate evaporation. The oxygen
content of the culture was monitored with the redox indicator resazurin.
2.4.2
Cell concentration via Minisart® filter
To harvest cells by filtering, culture fluid was taken from a culture within an anoxic glove box
using sterile and anoxic 3–10 ml plastic syringes (Omnifix®, Braun, Melsungen, Germany) and
filtered through a 0.2 µm pore size filter (Minisart®, Sartorius, Göttingen, Germany). Syringes were
made anoxic by filling with sterile reduced buffer, medium or water. The filter containing the cells
was then connected with the reverse side to a new syringe with the help of Luer Lock adapter. Cells
were flushed into an Eppendorf reaction tube and either applied in an activity assays or used for cell
counting.
2.4.3
Cell concentration via centrifugation
Centrifugation vials were filled with cell culture (max. 35 ml in 50 ml Falcon tubes or max. 11 ml
in 15 ml Falcon tubes) and centrifuged for one hour at 16°C and 5,000 g. Fifty percent of the
supernatant were removed in the anoxic glove box and the remaining solution was centrifuged for an
additional hour at 16°C and 5,000 g. The remaining supernatant was removed and the pellet
resuspended in buffer or fresh medium.
2.4.4
Density centrifugation using Nycodenz®
Density centrifugation with 80% (w/v) Nycodenz® (Nycodenz AG, Axis-Shield, Oslo, Norway)
solution was done for separation of whole cells from sediment particles. This procedure was modified
according to methods described previously (166-168). For the preparation of the solution Milli-Q
water was filled into a beaker and stirred with a stirring bar before solid Nycodenz® was added in
small amounts to facilitate the dissolving process and to avoid clumping. Finally the solution was
filled up to the required volume and filter-sterilized through a 0.2 µm filter. The 80% solution was
stored in a sterile Falcon tube at 4°C.
For the separation of cells from sediments, 10 g of sediment was filled into a 50 ml sterile
centrifugation tube. Thirty ml of a sterile 0.9% (w/v) NaCl solution was added and incubated for at
19
Material and Methods
least three hours, shaking horizontally at 450 rpm and room temperature. To further detach cells from
sediment particles, the tube was sonified for 1 minute in a sonification bath. Subsequently, the tube
was centrifuged for 6 min at 800 g, 4°C. The supernatant was transferred into sterile 15 ml
centrifugation tubes. The remaining sediments were stored at 4°C for a second extraction. A layer of 2
ml 80% (w/v) Nycodenz® was carefully injected to the bottom of the centrifugation tube below the 10
ml aqueous phase of the supernatant with a long needle. The tube was transferred to a centrifuge
without disturbing the Nycodenz®-liquid interface and centrifuged at 14,000 g, at 4°C, for 45 min.
Supernatant and Nycodenz® layer were transferred into a new 15 ml centrifugation tube without
disturbing the sediment pellet. The water-Nycodenz-mixture was thoroughly mixed by using a vortex
mixer until no phase separation was visible to lower the Nycodenz® concentration and to allow for
sedimentation of cells at the bottom of the centrifugation tube. Cells were pelleted by a third
centrifugation step at 14,000 g at 4°C for 15 min and the supernatant was discarded. To remove
remaining traces of Nycodenz®, the pellet was washed with 1 ml of a 0.9% NaCl solution and
centrifuged at 14,000 g at 4°C for 15 min. The dried pellet was resuspended in 100 µl 0.9% NaCl
solution. For parallel or subsequent extractions, the resuspended pellets were combined into one
centrifugation tube. Twenty µl cell suspension were withdrawn for cell counting (see section 2.3.3).
The cell pellet was obtained by centrifugation at 14,000 g at 4°C for 15 min and was used immediately
for activity assays, DNA extraction or was stored dry at -20°C for further usage.
2.5
Resting-cell activity assay
Methyl viologen and other viologens can be used in their reduced state as artificial electron
donors for reductive dehalogenase in an in vitro activity assay (105, 169). Titanium(III) citrate as
described above (section 2.3.1.1) was used to reduce viologens. In their reduced state viologens are
blue while in their oxidized state viologens are colourless. The catalytic activity of strain CBDB1 or
marine microcosms towards halogenated compounds was analysed by using whole non-disrupted cells
and methyl viologen or in specific cases benzyl and ethyl viologen as an artificial electron donor.
Figure 2-2: Principle of the viologen-based activity assay
20
Material and Methods
2.5.1
Preparation of solutions and cell suspensions for the activity assay
For the preparation of potassium acetate buffer 46 ml of a 2 M potassium acetate stock solution
and 4 ml of a 2 M acetic acid stock solution were filled into a 100 ml Schott bottle and adjusted to pH
5.8 with a potassium acetate solution or acetic acid (170). The solution was then filled to a volume of
100 ml with Milli-Q water and filtered through a 0.2 µm sterile filter. Methyl viologen was prepared
as a 10 mM solution (111). Methyl viologen solution and potassium acetate buffer were degased by
applying vacuum in the air lock of an anoxic chamber for 30 min and subsequently equilibrated in the
anoxic glove box for at least 24 hours prior to usage.
Halogenated compounds were dissolved in acetone to obtain a final concentration of 50 mM.
Titanium(III) citrate was used from a stock solution (see section 2.3.1.1). Cells were concentrated to at
least 1 x 107 cells ml-1 using either a rotary evaporator or a 0.2 µm filter (see section 2.4.1 and 2.4.2).
Twenty µl were removed from the whole cell suspension for cell counting (see section 2.3.3). The
protein concentration was calculated by using the cell numbers and protein amount determination by
Anja Kublik (personal communication) with 30 fg per cell. All solutions and cell suspensions were
prepared in an anoxic glove box.
The activity assay solution contained potassium acetate buffer (pH 5.8), titanium(III) citrate,
methyl viologen and halogenated electron acceptors (see Table 2-8). Halogenated electron acceptors
were added from a 50 mM stock solution in acetone. For every tested compound a separate reaction
mix was prepared in a glass reaction vial, sealed and equilibrated for at least 10 min before a catalyst
was added. The negative control contained acetone without electron acceptor and the assay solution.
The positive control contained 1,2,3-trichloro- or 1,2,3,4-tetrachlorobenzene.
Table 2-8: Activity assay solution for 10 ml
Compound
Anoxic H2O
Methyl- ethyl- or benzyl viologen
(10 mM in anoxic H2O)
Potassium acetate buffer pH 5.8
(1 M in anoxic H2O )
Titanium(III) citrate (~ 100 mM)
Electron acceptor (50 mM in acetone)
Anoxic H2O
2.5.2
added volume (ml)
7
1.25
Final conc. (mM)
1.25
1.25
125
~ 0.01
0.125
Ad 10
~2
0.625
Gas chromatography-based activity assay
For the GC-FID-based activity assay 80 µl cell suspension and 800 µl assay solution were filled
into 2 ml HPLC vials in an anoxic chamber, sealed with Teflon-lined rubber septa and incubated at
30°C. At time zero and after 1 to 15 hours of incubation the reaction was stopped by hexane
extraction. Extracts were analysed for dehalogenation products using a GC-FID (see section 2.6.1).
The activity was calculated from the amount of electron acceptor which reacted after one to fifteen
21
Material and Methods
hours compared to the amount of electron acceptor measured at time zero or from the amount of
products formed.
2.5.3
Photometer-based microtiter plate activity assay
The microtiter plate activity assay was set up in an anoxic chamber using a 96-round bottom well
glass microtiter plate (Zinsser Analytic, Frankfurt, Germany). Each plate contained negative controls
with cells and acetone but without an halogenated electron acceptor and positive controls with cells
and 1,2,3-trichloro- or 1,2,3,4-tetrachlorobenzene. A chemical control was set up to test for abiotic
reactions of the electron acceptors with compounds from the reaction mix. The chemical control
contained an electron acceptor, the reaction mix and cell-free media. It was measured either in a
separate plate or was included with three parallels on the same plate in which resting cell activity was
measured. Every electron acceptor was tested for activity with resting cells in at least three parallel
wells (see Figure 2-3). Twenty or forty µl of whole cell suspension with approximately
1 x 107 cells ml-1 and 200 µl assay solution were filled into the designated wells. The plate was
immediately sealed with a transparent PCR-foil seal (VWR, Dresden, Germany).
1
2
3
4
5
6
7
8
9
10
11
12
A
R
R
R
R
R
R
R
R
R
R
R
R
B
R
PC
E
E
E
E
E
E
E
E
E
R
C
R
PC
E
E
E
E
E
E
E
E
E
R
D
R
PC
E
E
E
E
E
E
E
E
E
R
E
R
NC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
R
F
R
NC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
R
G
R
NC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
AEC
R
H
R
R
R
R
R
R
R
R
R
R
R
R
Figure 2-3: Example for a microtiter well plate set-up with negative controls (NC), positive controls (PC),
electron acceptor, reaction mix and resting cells (E), abiotic electron acceptor controls (AEC) and reaction mix
without cells and electron acceptor (R). The latter were included to avoid any influence of oxygen traces
diffusing from the atmosphere of the tent below the transparent film into the plate wells, so that only the inner
wells of the plate were used for measurements.
The activity was measured photometrically by following the oxidation of methyl viologen at its
absorbance maximum of 578 nm (extinction coefficient: 9.78 mM-1 cm-1) with a microtiter plate reader
from Synergy HT, BioTek (Bad Friedrichshall, Germany). To monitor background absorbance
originating from a condensate formed on the plastic film, the absorbance at 450 nm was measured
additionally to off-set the absorbance at 450 nm with the absorbance measured at 578 nm. The initial
absorbance of the assay solution at 578 nm was adjusted to 1.5 to 3.5 with titanium(III) citrate. The
absorbance was monitored for 15 h at 30°C. Data were recorded using the Gen5™ program 1.06
(BioTek, Winooski, VT, USA) with the following program settings:
22
Material and Methods
Table 2-9: Settings for the photometric activity assay with the Gen5™ program
Parameter
Temperature set point
Start kinetic
Shake
Read
Setting
30°C,
Run 15 h, Interval 7.5 min
Medium for 3 s
(A)450, 578, 650
The specific activity was calculated from the slope of the linear part of measured curves (Figure
2-4). To obtain activity rates in nkat mg-1 of protein of strain CBDB1 cells a conversion factor
according to Tina Hölscher was used (170) and then recalculated according to new protein
determination by Anja Kublick, UFZ Leipzig of 30 fg protein per cell (personal communication).
∆
Spec. Act.
∗
∆
‐1∗
∗
‐1 ∗ ∗
∗
.
∗
∗ ‐1
∗ ‐1
Figure 2-4: Formula for the calculation of the specific activity of resting cells based on the decrease of methyl
viologen absorption. Ee-acceptor: extinction measured in a sample with electron acceptor and cells; Eblank:
extinction measured in negative controls without electron acceptor but with cells, t: extinction measuring time in
minutes, always a time in the linear range of the curve was chosen; V total: combined volume of reaction mix
and resting cell sample (µl); εMV: extinction co-efficient of methyl viologen (9.78 cm-1 mM-1); d: pathway of
light through the sample (cm); V sample: volume of the sample (µl), Nmv: number of methyl viologen molecules
consumed per halogen released, P: protein concentration (cell density (cells ml-1) x cellular protein content (mg
protein cell-1)). A value of 30 fg cell-1 was used as a cellular protein content. The factor 16.67 compensates for
the calculation from µkat to nkat and from minutes to seconds.
2.5.4
Heat inactivation of cells for activity assay
To investigate background activity detected in activity assays, cells were harvested in an anoxic
glove box and transferred into gas-tight crimp vials. The vials were incubated for thirty minutes in a
water bath, either at room temperature, at 60°C, or at 80°C. Cells were transferred to the anoxic glove
box and used for comparative activity assays.
2.5.5
Reversible inhibition of reductive dehalogenases
The reversible inhibition of dehalogenase activity with alkyl iodides was used to investigate i) the
background activity in the microtiter plate activity assay with strain CBDB1 and ii) the involvement of
a cobalamin-dependent reductive dehalogenase in marine sediment microcosms (169). Resting cell
suspensions were reduced with titanium(III) citrate (2 mM final conc.) in an anoxic glove box. Propyl
iodide was added from an acetonic stock solution to obtain a final concentration of 1 mM for cell
suspensions of strain CBDB1 or 10 µM for cell suspensions of marine sediment microcosms. The
same amount of acetone without propyl iodide was added to a negative control. The cell suspensions
were incubated for one hour in 2 ml HPLC vials, covered with aluminium. All vials were sealed with
Teflon-lined rubber stopper. After one hour of incubation at room temperature the aluminium foil was
removed from selected vials with propyl iodide and vials without propyl iodide and exposed to a 250
23
Material and Methods
watt light source for 30 min. Three different experimental set-ups, each in triplicates were compared:
a) Propyl iodide treated cell suspensions incubated in the dark without exposure to a light source, b)
propyl iodide treated cell suspensions exposed to light and c) controls incubated without alkyl iodide
exposed to light. All were tested for dehalogenase activity by the microtiter plate activity assay and/or
a GC-FID-based activity assay (see section 2.5).
2.5.6
Oxygen sensitivity tests with marine cultures and activity assay
Culture liquid was retrieved from cultures in an anoxic glove box using a 5 ml syringe
(Omnifix®-F, Braun AG, Melsungen, Germany) previously flushed with anoxic water. The cell
suspension was transferred into 2 ml HPLC vials, each time point in triplicates. Cells were stirred with
stirring bars to oxygenize the cell suspension and incubated at room temperature for 0.5, 1, 2 and 4
hours in the anoxic glove box and in a parallel set-up outside the anoxic glove box to expose the
samples to oxygen. After the incubation time vials incubated outside the anoxic glove box were
transferred into the anoxic chamber. Cells incubated the same time in the anoxic chamber and cells
exposed to oxygen were both used for setting up GC-FID-based activity assays. The samples were
incubated at 30°C for 12 hours in the activity assay solution and subsequently analysed for reaction
products.
2.5.7
Activity assays with cells from sediments
Sediments stored at 4°C from the Baffin Bay, from tidal flat sediments and from the Aarhus bay
were used for activity assays in HPLC vials. Sediments stored in gas-tight Schott bottles at 4°C were
introduced into the anoxic glove box and 0.5–3 g were transferred into 2 or 5 ml HPLC vials. The
reaction mix for activity assays was prepared with 1,2,3,4-tetrachlorobenzene, 2,4,6-tribromophenol,
1,2,3-trichlorobenzene or 1,2,4-dibromobenzene. The assay solution (see section 2.5.1) was added to
the sediments and the vials were incubated at room temperature or at 30°C for 12 to 60 hours. After
incubation the assay solution was transferred to a new glass reaction vial and the reaction was stopped
by extraction with hexane in a ratio of 1:2 assay solution to hexane. Alternatively, cells were extracted
from sediments using Nycodenz® and were set up with the activity assay as described above.
24
Material and Methods
2.6
Analytical methods
2.6.1
Gas chromatography
Brominated and chlorinated benzenes, chlorinated and fluorinated benzonitriles, chlorinated
thiophenes, anilines and pyridines from cultivation and resting cell activity assays were extracted from
0.5 ml samples with 1 ml of hexane. All compounds and reaction products except for benzene and
thiophene were analysed with a Shimadzu GC 14A equipped with a flame ionization detector and a
Permabond-FFAP capillary column (25 m, 0.25 mm i.d., 0.25 µm film thickness, Macherey & Nagel,
Düren, Germany). Generally, 1.5 µl samples were injected in splitless mode. Nitrogen (99.999%
purity) was used as carrier and make-up gas. The gas pressure settings were the following: Air 0.20 kg
cm2-1, hydrogen 0.6 kg cm2-1, nitrogen 0.65 kg cm2-1 as carrier gas and 1.00 kg cm2-1 as make up gas.
The temperature program started with an initial hold at 55°C for 1 min, an increase of temperature at a
rate of 6°C min-1 to 225°C with a final hold for 2 min. The injector temperature was 250°C and the
detector temperature 300°C.
Benzenes and thiophenes could not be detected with the same program as both reaction products
were covered by the hexane solvent peak. Therefore, these compounds were analysed from the
headspace of the reaction vial. Two hundred µl of the headspace were removed from the sealed
reaction vial using a gas-tight 200 µl glass syringe (Hamilton, Bonaduz, Switzerland) and transferred
directly into the injector of a HP5890II gas chromatograph equipped with a flame ionization detector
and a DB-5 column (30 m, 0.32 mm i.d., 0.25 µm film thickness, J&W Scientific, Köln, Germany).
Nitrogen (99.999% purity) served as carrier and make-up gas. The column temperature was 35°C
when the sample was injected. After 5 min the column temperature was increased at a rate of 5°C
minute-1 to 60°C and then at a rate of 30°C minute-1 to 300°C, with a final hold of 10 min at 300°C.
The injector temperature was 240°C and the detector temperature was 320°C.
To allow gas chromatographic analysis, bromophenols were acetylated with 50 mM acetic
anhydride in 120 mM sodium bicarbonate. Therefore, sodium bicarbonate and acetic anhydride were
added to 1 ml culture volume in a glass reaction vial, sealed and placed on a rotary shaker set to 350
rpm. The sample was mixed for 10 min at room temperature before extracting reaction products with 1
ml hexane. Derivatized bromophenols were analysed using a Hewlett Packard gas chromatograph
6890 equipped with a flame ionization detector and a HP-5 capillary column (30 m, 0.25 mm inner
diameter, 0.25 µm film thickness, Agilent Technologies, Böblingen, Germany). The temperature
program was as follows: initial hold at 55°C for one minute, the temperature then increased by 10°C
minute-1 until it reached 150°C and continued by an increase of 10° minute-1 to 230°C and then 30°C
minute-1 to 260°C with a final hold of 2 min. Injector and detector temperatures were both 250°C.
Injection was done splitless.
All compounds and most reaction products were identified and quantified by injecting single
reference compounds as standards in concentrations from 5 to 100 µM. For the benzene standard curve
25
Material and Methods
cultivation bottles with 60 ml anoxic water sealed with Teflon lined rubber septa were spiked with
benzene in concentrations between 5 and 100 µM. The detection limit for all compounds was 1–5 µM.
2.6.2
Ion chromatography
Transformation of brominated furoic acids and benzoic acids was analysed by taking 1 ml
samples of the cultures for measuring the bromide concentration using a DIONEX-ISC2000 Ion
Chromatography System equipped with an IonPacAS18/AG18 column (4 mm inner diameter, Thermo
Fisher Scientific, Bremen, Germany). Samples were measured according to the ISO 10304-2: 1995
protocol part 2 for the determination of bromide, chloride, nitrate, nitrite, ortho-phosphate and
sulphate in waste water.
2.6.3
NanoLC-ESI-MS/MS (LTQ-Orbitrap) analysis
For the analysis of expressed proteins by nano LC-ESI-MS/MS (LTQ-Orbitrap) of protein digests
strain CBDB1 was cultivated in 50 ml serum bottle flasks with mono- 1,2-, 1,3-, or 1,4-di- or 1,2,4tribromobenzene. When cultures were harvested for protein analysis they contained cell numbers from
2 to 4 x107 cells ml-1. Cells were obtained by filtering 30 ml of culture volume through a 0.2 µm filter
(see also 2.4.2). Subsequently cells were lysed, treated with DTT and iodoacetamide and digested with
trypsin as described previously (171). The obtained peptides were purified, desalted and concentrated
by ZipTip® pipette tips (Millipore, Eschborn, Germany). The peptides were reconstituted in 0.1%
formic acid for nanoLC-ESI-MS/MS measurements. The samples were trapped with 0.1% formic acid
in water and at flow rates of 15 µl min-1 (nanoAcquity UPLC column, C18, 180 μm x 2 cm, 5 μm,
Waters, Eschborn, Germany). The peptides were eluted after eight minutes onto the separation column
(nanoAcquity UPLC column, C18, 75 μm x 100 mm, 1.7 μm, Waters, Eschborn, Germany).
Chromatography was done with a nano-UPLC system (nanoAquity) and the solvents used for
chromatography (solvent A 100% water, solvent B 100% acetonitrile) contained 0.1% formic acid. For
the elution of the peptides a 6–20% solvent B gradient was applied over 90 min and continued with a
20–85% solvent B gradient over 60 min. A TriVersa NanoMate LC-coupler (Advion) was used for
coupling of the nano-UPLC system to the LTQ-Orbitrap XL mass spectrometer (Thermo Fisher
Scientific). The eluted peptides were scanned continuously at 300–1600 m/z, automatically switching
to MS/MS CID mode with normalized collision energy of 35% and an activation time of 30 ms on top
six peptide ions exceeding an intensity of 3,000. An exclusion mass width of ±4 ppm over 3 min
enabled dynamic exclusion. Obtained data were evaluated using the tandem mass spectrometry ion
search algorithms from the Mascot house server (v2.2.1). For the search, a taxonomy ID 255470
(Dehalococcoides mccartyi strain CBDB1) of NCBI nr (National Centre for Biotechnology
Information, Rockville, MD), tryptic cleavage and a maximum of two missed cleavage sites was
selected. Additionally carbamidomethylation at cysteines was given as a fixed and oxidation of
methionines as a variable modification. The peptide tolerance threshold was set at ±10 ppm and the
26
Material and Methods
MS/MS tolerance at ±0.8 Da and peptides were considered to be identified by Mascot when a false
positive probability of 0.05 (probability-based ion score threshold of 40) was obtained. For identified
peptides and their respective proteins the protein score and the emPAI (exponentially modified protein
abundance index (172)) were calculated by Mascot. A higher protein score corresponds to a more
confident match between the ion score and the amino acid sequence of the protein while the emPAI
allows an approximate quantitation of the protein by dividing the number of detected peptides by the
number of expected peptides and subsequent exponential modification of the calculated value.
2.7
Molecular methods
2.7.1
Chloroform-based DNA extraction
DNA was extracted according to a modified protocol described by Miller and colleagues (173)
from crude cell pellets obtained via Nycodenz® density centrifugation from 40 g of sediments (see
section 2.4.4). Cells were resuspended in 1.35 ml extraction buffer (100 mM Tris-HCl, 100 mM
EDTA, 100 mM sodium hydrogen phosphate, 1.5 M NaCl, pH 8.0) and transferred to a fresh 2 ml
centrifugation vial. Five µl of fresh Proteinase K (20 mg ml-1) was added and carefully mixed by
gently flicking the tube. The tube was incubated at 37°C for 30 min and carefully mixed every ten
minutes. 150 µl of 20% (w/v) SDS were added, incubated at 65°C for 2 hours and mixed by inversion
of the tube every fifteen minutes. After incubation the solution was centrifuged for 10 min at 6,000 g,
room temperature and the supernatant was transferred to a fresh sterile 2 ml centrifugation vial. This
step was repeated until the supernatant was clear. To the crude extract an equal volume of
chloroform-containing 4% (v/v) isoamyl alcohol was added and gently mixed by inversion. The
aqueous phase was separated from the chloroform phase by centrifugation at 16,000 g 10 min at room
temperature. The upper aqueous phase was transferred with a cut wide bored 1000 µl pipette tip (~3
mm opening) while leaving the interface undisturbed. The DNA was precipitated by adding 0.6 ml of
isopropanol to one ml of aqueous phase (ratio of 0.6:1). Isopropanol was mixed thoroughly with the
aqueous phase and incubated for one hour at room temperature to precipitate DNA. The tubes were
centrifuged for 20 min at 16,000 g for 20 min at room temperature. After removing the supernatant the
pellet was washed with 500 µl cold 80% (v/v) ethanol and centrifuged for 10 min at 16,000 g and
room temperature. The ethanol was removed while leaving the pellet undisturbed. The pellet was airdried for one hour in the dark and resuspended in 30 µl PCR grade H2O or in 0.1 x concentrated TEbuffer. The DNA concentration and quality was measured. In case of remaining protein
contaminations the DNA was additionally purified with the Wizard® SV Gel and PCR Clean-Up
System kit (Promega, Madison, WI, USA) and eluted with 30 µl PCR grade H2O. The DNA was
stored at -80° for further usage.
27
Material and Methods
2.7.2
DNA extraction with the FastDNATM Spin Kit for Soil
DNA was extracted from 0.5 g of sediments with the FastDNATM Spin Kit for Soil (Biomedicals,
Eschwege, Germany) according to the manufactures instructions in combination with the FastPrep
FP120 cell disrupter (Thermo Fisher Scientific, Waltham, Massachusetts, USA).
2.7.3
Whole genome amplification
Whole genome amplification of SAG-C11 was done using the Phi29 whole genome amplification
kit (GE Healthcare, Chalfont St Giles, Buckinghamshire, Great Britain) according to the instruction of
the manufacturer.
2.7.4
Determination of DNA concentration and purity
The concentration and purity of nucleic acids were determined by measuring the absorbance at
260 nm and 280 nm with a NanoDrop ND-1000 spectrophotometer. Additionally the concentration of
nucleic acids was estimated by comparing the amplified fragments with defined fragments of
commercially available DNA ladder.
2.7.5
Primer combinations and expected products
Dissimilatory sulphite reductase (dsr) related genes were identified in the Chloroflexi single
amplified genome C11 (SAG-C11). SAG-C11 was annotated in a parallel project by Dr. Kenneth
Wasmund. To investigate the distribution of dsr in marine sediments, primers were obtained from
Eurofins MWG Operon (Ebersberg, Germany) and diluted with PCR grade water to obtain a stock
solution with a concentration of 100 pmol µl-1. For PCR reactions, primers were diluted to
concentrations of 5 or 10 pmol µl-1. Primers for long range PCR of 3.9 to 10.1 kb were purchased with
a phosphorothioate 3 prime end modification to protect primers from exonuclease activity of the
phusion DNA polymerase during long amplification cycles (174). Primers dsr1F-DHC-2-pt and dsr4rC11 (Table 2-10) were based on the primers DSR4R and DSR1F used by Wagner and colleagues
(151). They were modified according to Chloroflexi SAG-C11. All other primers were based on the
sequence of SAG-C11 and degenerated positions were introduced after comparison of SAG-C11
sequences with BlastN hits from the NCBI nr database by aligning with these sequences using
MUSCLE implemented in Mega 6.0 (175).
28
Material and Methods
Table 2-10: Primer combinations used for the amplification of dsr-related genes from sediments (the
phosphorothioate modification is indicated in small letters). For several fragments different primer combinations
were used yielding fragments with a similar size.
type
Name
Sequence 5’ 3’
fw
rv
rv
rv
rv
rv
rv
fw
rv
rv
rv
fw
rv
fw
rv
fw
rv
fw
rv
fw
rv
rv
rv
rv
rv
rv
fw
rv
rv
rv
rv
rv
rv
fw
fw
rv
rv
rv
rv
rv
rv
fw
rv
Dsr1F-DHC-2-pt
SAG-D_Glut
SAG-D_Glut_plus
SAG-D_Glut-pt
SAG-D_Glut_v2
SAG-D_Glut_v3
SAG-D_Glut_v4
Dsr1F-DHC-2-pt
SAG-D_UropF1
SAG-D_UropF2
SAG-D_UropF3
C11_luxr1_out1
SAG_D_gammaF_C
Dsr1F-DHC-2-pt1
SAG_D_gammaF_A
Dsr1F-DHC-2-pt2
SAG_D_gammaF_B
Dsr1F-DHC-2-pt3
SAG_D_gammaF_C
C11_luxr1_out1
Dsr4R-C11
Dsr4R-C11plus
Dsr4R-C11plus2
Dsr4R-C11plus3
Dsr4R-C11plus4
Dsr4R-C11plus5
C11_luxr1_out2
Dsr4R-C11
Dsr4R-C11plus
Dsr4R-C11plus2
Dsr4R-C11plus3
Dsr4R-C11plus4
Dsr4R-C11plus5
C11_luxr1
C11_luxr1_deg
Dsr4R-C11
Dsr4R-C11plus
Dsr4R-C11plus2
Dsr4R-C11plus3
Dsr4R-C11plus4
Dsr4R-C11plus5
Dsr1F-DHC-2-pt
Dsr4R-C11
GATTACSCACTGGAAACAtg
TTGGCATTAATCATAAGAC
TTGGCATWAAYCATMAKAC
TTGGCATTAATCATAAGac
TACCTGTAACCGAACCga
CTTTTTCGAGTGGCATCcg
ATTGCTGTTCCTCCAGat
GATTACSCACTGGAAACAtg
AGGGAAAGGTCTATCTGgt
TGATAACAGTAAAGGGCct
AAAGGTGGTGACCCTTTTgt
CTCTAGAACTTTTCATCCG
AAGCCATCCTCATCAASyt
GATTACSCACTGGAARCAyg
AAGCCATCCTCRTCMASyt
GATTACACACTGGAARCAyg
AAGCCATCCTCATCMASyt
GATTACACACTGGAATCAyg
AAGCCATCCTCATCAASyt
CTCTAGAACTTTTCATCCG
GTAAAGCAATTGGCACA
GTRWAGCARTTRSCRCA
GTAAAGCAATTRSCRCA
GTAAAGCAATTGGCRCA
GTAAAGCAATTRGCRCA
GTAAAGCAATTGSCRCA
GCCCACCATGTGTATTCC
GTAAAGCAATTGGCACA
GTRWAGCARTTRSCRCA
GTAAAGCAATTRSCRCA
GTAAAGCAATTGGCRCA
GTAAAGCAATTRGCRCA
GTAAAGCAATTGSCRCA
GAACTTATCTTGGCTCTG
GAACTTATCTTGRCYCWG
GTAAAGCAATTGGCACA
GTRWAGCARTTRSCRCA
GTAAAGCAATTRSCRCA
GTAAAGCAATTGGCRCA
GTAAAGCAATTRGCRCA
GTAAAGCAATTGSCRCA
GATTACSCACTGGAAACAtg
GTAAAGCAATTGGCACA
29
Targeted
gene/s
Size (kb)
dsrABDNCMK,
cobA/hemD,
hemC, hemA
10.185
10.185
10.185
10.064
9.913
9.376
dsrABDNCM,
cobA/hemD
8.108
8.063
7.854
luxR, dsrABDN
4.687
dsrABDNC
3.896
dsrABDNC
3.896
dsrABDNC
3.896
luxR, dsrAB
2.589
luxR, dsrAB
2.667
luxR, dsrAB
2.502
dsrAB
1.978
Material and Methods
For the amplification of fragments cloned into the commercially available vectors pJET 1.2 and
pGEM T-easy specific primers binding within the sequence of the vector have been used (see Table
2-11). For testing marine dehalogenating microcosms for cross-contamination with strain CBDB1
primers targeting the reductive dehalogenase homologous gene cbdbA1453 were used (Table 2-12).
Table 2-11: Primers for the amplification of inserts from vectors using the flanking regions of the vector as
primer targets
combination
1
Name
pJET 1.2 fw
pJET 1.2 rv
Sequence 5’ 3’
CGACTCACTATAGGGAGAGCGGC
AAGAACATCGATTTTCCATGGCAG
2
pGEM T-easy M13 fw
pGEM T-easy M13 rv
GTAAAACGACGGCCAGT
GCGGATAACAATTTCACACAGG
Table 2-12: Primers used for testing for contamination by strain CBDB1 by amplification of cbdbA1453
Sequence 5’ 3’
AATCTCTCGAGGGCACTC
CCAGAGGGCTGGTAAGG
type
fw
rv
Name
cbdbA1453_f
cbdbA1453_r
2.7.6
Polymerase chain reaction
Polymerase chain reactions were carried out in an Eppendorf Mastercycler® Personal (Eppendorf,
Hamburg, Germany). For long range PCRs the Phusion®High-Fidelity DNA polymerase master mix
with HF buffer (New England Biolabs, Ipswich, Massachusetts, USA) was used. The final primer
concentration in the PCR reaction mix was 400 nM. The concentration of the template was typically
~10 ng µl-1. One µl template was added to a PCR reaction with a total volume of 25 µl. SAG-C11
DNA previously amplified using a Phi29 whole genome amplification kit (see section 2.7.3) was used
as a positive control for the amplification of dsr-related genes from sediments. For shorter fragments
up to 1.8 kb the Taq DNA polymerase (New England Biolabs, Ipswich, Massachusetts, USA) was
used. The elongation time depended on the used polymerase and the size of the amplified fragment.
The binding temperature of the applied primers was estimated according to the GC content and
optimized by a temperature gradient PCR.
Table 2-13: PCR reaction components and concentrations
Component
PCR-buffer 1x
MgCl2
Forward primer 10 µM
Reverse primer 10 µM
dNTPs (2.5 mM each)
DNA-polymerase
DNA template
H2O add to a total volume of
Taq DNA polymerase
(NEB)
2.5 µl
1.5 mM
0.4 µM
0.4 µM
250 µM
0.025 U µl-1
1 µl
25 µl
30
Phusion™ High Fidelity DNA
polymerase (NEB)
12.5 µl
1.5 mM
0.4 µM
0.4 µM
200 µM
0.02 U µl-1
1 µl
25 µl
Material and Methods
2.7.6.1 PCR temperature programs
The following programs were used for the amplification of DNA fragments from DNA isolated
from marine sediments (Table 2-14 to Table 2-16).
Table 2-14: PCR temperature programs for 1.8 and 2.5 kb fragments.
Fragment
Polymerase
Initial denaturation
Denaturation
Annealing
Elongation
Cycles
Final elongation
Hold
1.8 kb
Taq
Temperature
98°C
98°C
54°C
72°C
72°C
4°C
Time
15 sec
10 sec
20 sec
45 sec
35
5 min
∞
2.5 kb
Phusion
Temperature
98°C
98°C
54°C
72°C
Time
15 sec
15 sec
20 sec
45 sec
35
72°C
4°C
5 min
∞
Table 2-15: PCR temperature program for a touch-down PCR for a 2.5 kb fragment and degenerated primers
C11_luxr1_deg and Dsr4R-C11 and the Phusion Polymerase
Step
Initial denaturation
Denaturation
Annealing
Elongation
Cycles
Denaturation
Annealing
Elongation
Cycles
Final elongation
Hold
Temperature
98°C
98°C
60°C
-0.5°C
72°C
Time
15 sec
10 sec
20 sec
45 sec
12
10 sec
20 sec
45 sec
28
5 min
∞
98°C
54°C
72°C
72°C
4°C
Table 2-16: PCR temperature program for a 3.9 kb DNA fragment amplified with the Phusion DNA polymerase
Step
Initial denaturation
Denaturation
Annealing
Elongation
Cycles
Denaturation
Annealing
Elongation
Cycles
Final elongation
Hold
Temperature
98°C
98°C
58°C
72°C
Time
30 sec
10 sec
20 sec
4 min
30
98°C
58°C
72°C
10 sec
20 sec
5 min
10
72°C
4°C
10 min
∞
31
Material and Methods
The following PCR program was used to test marine microcosms for the presence of
contaminations by strain CBDB1 (Table 2-17).
Table 2-17: PCR temperature program for the amplification of the cbcb1453 of strain CBDB1 using the Hot
start Taq polymerase (NEB).
Step
Initial
denaturation
Denaturation
Annealing
Elongation
Cycles
Final elongation
Hold
2.7.7
Temperature
95°C
Time
15 min
95°C
59°C
72°C
30 sec
30 sec
1 min
35
10 min
∞
72°C
4°C
Agarose gel electrophoresis
For agarose gel electrophoresis 1% (w/v) agarose was dissolved in TAE buffer with a 0.5 x final
concentration (20 mM Tris-Acetat, 0.5 mM EDTA, pH 8.3). As running buffer TAE buffer with a 0.5
x final concentration was used as a running buffer. Separation of DNA fragments was done at 60–90
V. Agarose gels were stained with ethidium bromide and DNA fragments were visualized using an
UV-light detector (BioRad Gel Doc™ XR+Imager, Munich, Germany). The following DNA ladder
was used to estimate size and concentration of the amplified PCR products: MassRuler High Range
DNA Ladder (Thermo Scientific, Waltham, Massachusetts, USA).
2.7.8
Elution of DNA from agarose gels
PCR amplified fragments were separated on a 1% agarose gel and visualized by ethidium bromide
staining and UV-light. Gel pieces which contained DNA fragments with the required size were
excised with a scalpel. Gel pieces were purified using the Wizard® SV Gel and PCR Clean-Up
System kit (Promega, Madison, WI, USA). When PCR product amounts were very low, up to10 gel
pieces with DNA products of the same size were combined before gel purification. The purification of
PCR products via the PCR product wizard was done according to the manufacturer’s
recommendations. The PCR products were eluted in 30 to 50 µl of PCR grade water.
2.7.9
DNA and plasmid preparation and purification
Plasmids were purified with the NucleoSpin® Plasmid Kit (Macherey-Nagel, Düren, Germany)
according to the manufacturer’s instructions.
32
Material and Methods
2.7.10
Addition of an poly adenosine tail
Prior to cloning, DNA fragments obtained by the Phusion DNA polymerase were incubated with
deoxyadenosine triphosphate and Taq polymerase to add an additional adenosine overhang to the three
prime for TOPO TA cloning.
2.7.11
TA cloning
DNA fragments containing an additional adenosine overhang were ligated in the pGEM®-T-easy
vector for 12 hours at 4°C using the pGEM®-T-easy Vector System (Promega, Madison, WI, USA)
according to the manufactures instructions.
Table 2-18: Blunt end PCR products were incubated for 20 minutes at 72°C with following components
Component
Buffer (10x)
Taq Polymerase
dATPs (10 mM)
Template
Volume in µl
0.5
0.5
1
3
Table 2-19: Reaction mix for ligation
Component
Buffer
Vector
Ligase
Template
2.7.12
Volume in µl
2.5
0.5
0.5
1.5
Blunt end cloning
For blunt end cloning of fragments obtained with the Phusion DNA polymerase were ligated into
the pJET 1.2 blunt end cloning system (ThermoScientific, Waltham, Massachusetts, USA) according
to the manufactures instructions. Vector and insert were ligated for 30 min at 37°C.
2.7.13
Transformation
Clone libraries were constructed by transformation of ligation products into E. coli K12 cells
(NEB C2987, New England Biolabs, Ipswich, Massachusetts, USA). Cells were thawed on ice for ten
minutes. Five µl plasmid DNA was added to 50 µl competent cells and gently stirred with the pipette
tip. The cells were incubated for 30 min on ice. Subsequently, the cells were exposed for 30 s to 42°C
and then incubated 5 min on ice. After this 950 µl of SOC outgrowth medium (New England Biolabs,
Ipswich, Massachusetts, USA) were added and the cells were incubated at 37°C for one hour at 250
rpm. The mix was plated on selective LB-agar plates (80 µg ml-1 X-Gal, 100 µg ml-1 ampicillin).
2.7.14
Colony PCR
After ligation of specific fragment into the pGEM®-T-easy vector or the pJET 1.2 vector
transformation of E.coli cells and identification of positive clones with the blue white screening, the
33
Material and Methods
plasmid insert was re-amplified with the vector-specific primers M13 from pGEMT easy and pJET
(see Table 2-11) with an annealing temperature of 55°C. White colonies were resuspended in 100 µl
sterile water, incubated for 2 h at 37°, mixed on a Vortex mixer and centrifuged for 1 min at 6,000 rcf.
The supernatant was applied into the reaction mix and the solution was stored at -20°C.
2.7.15
Screening and sequencing of inserts
White colonies were picked, suspended in PCR grade water and screened for the correct insert
size by PCR and agarose gel electrophoresis using M13 forward and reverse vector primer or pJET 1.2
fw and rv primer (Table 2-11). For sequencing PCR fragments obtained via colony PCR were
separated on an agarose gel. The obtained fragment was purified using the PCR purification wizard
(Promega, Fitchburg, Wisconsin, USA). For sequencing at the GATC European Custom Sequencing
Centre (Köln, Germany), 5 µl of a 20–80 ng µl-1 PCR amplified DNA fragment were mixed with 5 µl
of vector primer (5 pmol µl-1) (see Table 2-11 page 30). For sequencing, plasmids were purified with
the NucleoSpin® Plasmid Kit (Macherey-Nagel, Düren, Germany). For sequencing, plasmids were
used at 80–100 ng µl-1. The full-length sequence of several selected clones was sequenced by using
vector primer and internal primers (see Table 2-20). For internal primers a sequence with a length of
19 to 21 bases and a GC content of 40–60% was selected in a position of approximately 150 bases
before the 3´ end of the sequenced fragment. For obtaining full-length sequences of long-range PCR
fragments, primers were selected based on the sequences amplified from sediments.
Table 2-20: The following primers were used for internal sequencing of inserts
Primer name
PW_dsrB_75
PW2_dsrB_75
PW_dsrB_11
PW2_dsrB_11
PW_dsrB_20
PW2_dsrB_20
PW3_dsrA_11 u 20
PW_dsrA_290
PW2_dsrA_290
PW3_dsrB_290
PW4_cobyrinic_290
PW_dsrA_252
PW2_dsrA_252
PW3_dsrB_252
PW4_cobyrinic_252
PW_dsrA_265
PW2_dsrA_265
PW3_dsrD_265
PWb_dsrA_285
PW2_dsrA_285
PW3_dsrB_285
PW5_cobyrinic_285
PW_dsrA_271
Sequence 5’–3’
ACCTCAGATAGCCATCACAG
GTTGATACAGTGCATACATCT
TCCTGAGGCATCTGTTGCCG
ATCGTAAGGTGGCTCCATCT
CCGTATCGAATCGACACTTA
TTCTCATCCCAGTTCAGGGC
AGTTCCTTAGCTGCCGGTT
TGCCCCAATGACTGTGTTGC
CAGTTGAGGAGAGCGCTTTG
TCACCGACCAGCATATTGAT
ATCGACAATATTTGCCATC
CGATGCCCTGACTGATGAGG
CACCATATACTGAGTTGAAAG
AAGGCGATGGCATCTCCATC
GCTATGGTAACAGGTTATCA
AGGATGCCCAAGGCACTTAG
GGCTATCTGAGGTTTACCAGC
GCTGGTTGATAAGGCAATAG
TATCCAGAATGAAGTTATCA
TTAAGCGTCGATTCTGTACGG
GACTTCGACGCAGTTCAGG
GCATATAGCGCAGGCTATT
CTCAACCTGAAGCCAGAGGA
34
Target gene
dsrB
dsrB
dsrB
dsrB
dsrB
dsrB
dsrA
dsrA
dsrA
dsrB
dsrN
dsrA
dsrA
dsrB
dsrN
dsrA
dsrA
dsrD
dsrA
dsrA
dsrB
dsrN
dsrA
Material and Methods
Primer name
PW2_dsrA_271
PW3_dsrB_271
PWb_dsrA_215b
PW2_dsrA_215
PW3_dsrB_215
PW4_cobyrinic_215
2.8
Sequence 5’–3’
GTTGAGTATTGATACAGTGCG
CAACTACACAGTTCAAGTGG
GTTCTACTCTACCAAAGCTCT
TATAGAACCATGCTTCACC
CGTGACAACCTTCCGGAGT
GTAATGCTTGACCACGTCT
Target gene
dsrA
dsrB
dsrA
dsrA
dsrB
dsrN
Bioinformatic tools
Sequences obtained from the GATC sequencing service were evaluated for their quality using
Chromas Lite 2.1.1 (Technelysium Pty Ltd, South Brisbane, Australia). Functional annotation of genes
was done by Blast (Basic Local Alignment Search Tool (176)) and the NCBI non-redundant database
(nr) supplemented with the single-cell genome sequence of Dehalococcoidia C11. Sequences were
aligned using the muscle program (177). Sequences obtained with internal primers from long range
PCR fragments were assembled with the Sequencher program (Gene Codes Corporation, Ann Arbor,
USA). For these assembled sequences initial annotations were done using RAST 2.0 (178) and
Artemis (179).
For the construction of phylogenetic trees, DNA or amino acid sequences of functional genes
were aligned by using Muscle implemented in the Mega 6.0 program and nucleotide or amino acid
sequences retrieved from the NCBI non-redundant database. The obtained alignment was used to
construct phylogenetic trees using the Maximum Likelihood (180), Neighbor Joining (181) and
Minimum evolution (182) algorithm implemented in Mega 6.0 (175) and compared for achieved
topology. Bootstrap analysis was done with 1000 repetitions to test the stability of the tree topology.
Pairwise deletion was selected for gaps and missing data treatment and the p-distance method was
used for evolutionary distance calculation (183). The visualization of topologies were optimized with
the online tool “iTOL” (184, 185).
2.9
Electron density distributions for halogenated aromatics
To calculate the chemical parameters geometry optimization of halogenated aromatics were
carried out. Density Functional Theory level B3LYP/6-31G(d,p) was used to obtain a minimum
energy structure for each molecule and correct ground-state geometries were confirmed by frequency
analysis. The Gaussian 09 revision C.0149 (186) was used to carry out Mulliken (187), Hirshfeld
(188) (keyword IOP(6/79=1)) and NBO (189) population analysis. The analyses were done together
with D. Wondrousch, Department Ecological Chemistry, UFZ Leipzig.
35
Results
3
Results
3.1
Respiration and growth of Dehalococcoides mccartyi strain CBDB1 with
halogenated aromatic and heteroaromatic electron acceptors
Dehalococcoidia are ubiquitously distributed in marine sediments and reductive dehalogenase
homologues genes have been detected in non-contaminated marine sediments (60, 61). Because so far
all attempts to cultivate Dehalococcoidia from marine sediments remained unsuccessful, D. mccartyi
strain CBDB1 was used as a model organism for Dehalococcoidia to investigate potential electron
acceptors for respiration beyond known halogenated contaminants from anthropogenic sources. For
this strain CBDB1 was cultivated with halogenated electron acceptors, containing different halogen
and non-halogen substituents or heteroatoms: The compounds were selected based on structural
considerations to elucidate chemical parameters influencing reductive dehalogenation. The
information might help to clarify which functional groups of natural haloorganic molecules and
haloorganics from anthropogenic sources allow for reductive dehalogenation and growth of
organohalide-respiring members of the class Dehalococcoidia and may thereby help future attempts
for cultivation of Dehalococcoidia from marine sediments. In addition, the selected molecules contain
structures present in currently used halogenated xenobiotics and may therefore also contribute to a
better understanding of the role of Dehalococcoidia in fate prediction of halogenated xenobiotics in
anaerobic environments.
3.1.1
Chlorinated anilines as electron acceptor
To investigate the influence of amino substituents onto reductive dehalogenation, and growth with
halogenated anilines, D. mccartyi strain CBDB1 was cultivated in triplicates with 2,3-, 2,4- and
2,6-dichloroanilines. For this, culture medium was inoculated with a 10% inoculum of strain CBDB1
and dichloroanilines were added to an initial concentration of approximately 100 µM. After 70 days of
incubation at 30°C, 100 µM 2,3-dichloraniline were transformed. Additional 200 µM 2,3dichloroaniline were transformed after 104 days of cultivation. (Figure 3-1, left). In chemical controls
with 2,3-dichloroaniline but no inoculum, no transformation of 2,3-dichloroaniline was observed. In a
second transfer with 2,3-dichloraniline and an inoculum of 10%, approximately 87 µM of
2,3-dichloroaniline were transformed within 14 days in six parallel cultures. Additional 168 µM 2,3dichloroaniline were transformed within 43 days. In cultures grown with 100 µM 2,4- or 2,6dichloroaniline no transformation was detected (Figure 3-1; left). In chemical controls with 2,4- or
2,6-dichloroaniline, no transformation was detected.
Triplicate cultures with 2,3-dichloroaniline present as electron acceptor were inoculated with
starting concentration of 1.5 x 106 to 3.2 x 106 cells ml-1. Cell numbers increased constantly (5.2 x 106
cells ml-1 after 35 days and 1.10 x 107 cells ml-1 after 56 days) and reached final cell numbers of 1.5 to
36
Results
2.5e+7
300
2.0e+7
-1
cell growth (cells ml )
concentration (µM)
250
200
150
100
1.5e+7
1.0e+7
5.0e+6
50
0
0.0
0
20
40
60
80
100
120
140
CA ,4-DCA ,6-DCA
2,3-D
2
2
electron acceptor
time (days)
Figure 3-1: Transformation of chlorinated anilines and concomitant growth of D. mccartyi strain CBDB1. Left:
Transformation of dichloroanilines in cultures of strain CBDB1 (○ – 2,3-dichloroaniline, ● – 2,4-dichloroaniline;
 – 2,6-dichloroaniline). Approximately 200 µM 2,3-dichloroaniline was added after 95 days to the cultures set
up with 2,3-dichloroaniline. Right: Cell numbers of D. mccartyi strain CBDB1 in cultures with one of the tested
chlorinated anilines immediately after inoculation (black bars), and cell numbers after 140 days of incubation
(grey bars). Shown are mean values of triplicate cultures ± SD.
2.8 x 107 cells ml-1 after 140 days of cultivation (Figure 3-1, right). This corresponds to a cell growth
of 0.6 x 1014 cells mol-1 of halogen released. Cultures with 2,4-dichloroaniline as electron acceptor
were inoculated with 1.8 to 2.4 x 106 cells ml-1. After a cultivation period of 140 days, cell numbers
had decreased to 4.3 to 5.0 x 105 cells ml-1 (Figure 3-1, right). Similarly, in cultures with 2,6dichloroaniline as electron acceptor, cell numbers had decreased from 1.2 to 2.1 x 106 cells ml-1
initial cell number to 0.7 to 1.1 x 106 cells ml-1 after 140 days of incubation (Figure 3-1, right). In the
second transfer with six parallel cultures of strain CBDB1 and 2,3-dichloroaniline as electron acceptor
with initial cell numbers of 4–10 x 105 cells ml-1, growth was detected in all cultures. Cell numbers
reached 2–4 x 107 cells ml-1 after 83 days of incubation, corresponding to a growth yield of 0.9 x 1014
cells mol-1 halogen released.
3.1.2
2,3-Dichloronitrobenzene as electron acceptor
To investigate the influence of nitro substituents onto reductive dehalogenation, and growth with
halogenated nitrobenzenes, 2,3-dichloronitrobenzene was tested as an electron acceptor for D.
mccartyi strain CBDB1. However, 2,3-dichloronitrobenzene reacted abiotically with the reducing
agent during the equilibration time of 12 hours to 2,3-dichloroaniline. One culture transformed 70 µM
2,3-dichloroaniline after 28 days and additional doses of 110 µM 1,2-dichloronitrobenzene (which
reacted to 2,3-dichloroaniline) after 49 days (see 3.1.1). Initial D. mccartyi strain CBDB1 cell numbers
of 2 x 106 cells ml-1 increased 10-fold to 1.7 x 107 cells ml-1 after 56 days of incubation. A second
37
Results
culture transformed 70 µM 2,3-dichloroanilines after 77 days of incubation and cell numbers increased
from initial cell numbers of 2.9 x 106 to 9.8 x 106 cells ml-1.
3.1.3
Dehalogenation of and growth with 2-chloro-6-fluorobenzonitrile
To investigate growth with halogenated benzonitriles and the influence of the cyano group onto
reductive dechlorination and defluorination, strain CBDB1 was cultivated in triplicates with
2-chloro-6-fluorobenzonitrile. 2-Chloro-6-fluorobenzonitrile was added in crystals to the medium.
Bottles were incubated overnight to equilibrate before inoculation was done with strain CBDB1
previously
grown
with
1,2,3-trichlorobenzene
as
electron
acceptor.
Initial
2-chloro-6-
fluorobenzonitrile concentrations were approximately 260 µM. After 70 days 2-chloro-6fluorobenzonitrile was completely transformed to 2-fluorobenzonitrile (Figure 3-2). The product was
identified by parallel cultivation of 2,6-dichlorobenzonitrile and comparison of measured retention
times of reaction products. Defluorination to benzonitrile was not observed. In chemical controls with
2-chloro-6-fluorobenzonitrile no transformation products were detected. A second transfer of three
cultures was inoculated with a volume of 10% of strain CBDB1 culture pregrown on
2-chloro-6-fluorobenzonitrile and contained an initial concentration of 467 ± 128 µM of 2-chloro-6fluorobenzonitrile. Only one of three cultures containing 532 µM 2-chloro-6-fluorobenzonitriles
showed transformation of 155 µM 2-chloro-6-fluorobenzonitrile to 2-fluorobenzonitrile within 111
days of cultivation.
In the first transfer three serum bottles containing culture medium and 2-chloro-6-fluorobenzonitrile were inoculated with starting concentration of 6.8–7.5 x 106 cells ml-1. Cell numbers
8e+7
300
6e+7
200
4e+7
100
2e+7
concentration (µM)
-1
400
cell growth (cells ml )
increased constantly over time and reached final cell numbers of 4.7 to 6.9 x 107 cells ml-1 after 62
0
0
0
20
40
60
80
time (days)
Figure 3-2: Transformation of 2-chloro-6-fluorobenzonitrile (●) and concomitant growth of D. mccartyi strain
CBDB1 (grey bars). Shown are means of triplicate cultures from the first transfer ± SD.
38
Results
days of cultivation (Figure 3-2), corresponding to a cell growth of 2.3 x 1014 cells mol-1 of halogen
released. In the second transfer with three parallel cultures with initial cell numbers of 2.1 x 106 to
2.9 x 106 cells ml-1, growth was detected only in the culture, which transformed 2-chloro-6-fluorobenzonitrile. For this culture, cell numbers had increased from 2.4 x 106 cells ml-1 to 3.3 x 107 after
111 days of incubation, corresponding to a growth yield of 0.9 x 1014 cells mol-1 halogen released.
3.1.4
Chlorinated pyridines as electron acceptor
Strain CBDB1 was cultivated with chlorinated pyridines to investigate the influence of nitrogen
heteroatoms onto reductive dehalogenation, and growth with halogenated pyridines. For this purpose,
cultures were set-up in triplicates containing a 10% inoculum of strain CBDB1 and either 2,3- or
2,6-dichloropyridine as electron acceptor. The concentration of 2,3-dichloropyridine decreased from
48 ± 5 µM to 33 ± 0.2 µM after 97 days of incubation, however, no transformation products were
detected (Figure 3-3, left). For cultures with 2,6-dichloropyridine, the concentration of
2,6-dichloropyridine decreased slightly to 17 ± 1.3 µM after 97 days of incubation but no
transformation products were observed (Figure 3-3, left). In a second set-up with the same culture and
inocula volumes, 2,3-dichloropyridine and 2,6-dichloropyridine were added with initial concentrations
of 35 ± 4 µM and 40 ± 3 µM, respectively. The concentration of the two tested dichloropyridines did
not decrease over 60 days and no transformation products were detected.
Initial cell numbers of cultures incubated with approximately 48 µM 2,3-dichloropyridines were
1.5 to 2 x 106 cells ml-1. After 97 days of cultivation 0.9 to 2.3 x 106 cells ml-1 were detected (Figure
100
4e+6
3e+6
-1
cell growth (cells ml )
concentration (µM)
80
60
40
2e+6
1e+6
20
0
0
0
20
40
60
80
100
CPy
2,3-D
CPy
2,6-D
electron acceptor
time (days)
Figure 3-3: Transformation of chlorinated pyridines and cell numbers of D. mccartyi strain CBDB1. Left:
Concentration of 2,3-dichloropyridine (○) and 2,6-dichloropyridine (●) during cultivation of strain CBDB1.
Right: Cell numbers of strain CBDB1 in cultures with one of the chlorinated pyridines after inoculation (black
bars) and after 97 days of incubation (grey bars). Shown are means of triplicate cultures ± SD.
39
Results
3-3, right). Cultures with 2,6-dichloropyridine had initial cell numbers of 1.1 to 2.2 x 106 cells ml-1.
After 97 days of cultivation 1.9 to 2.1 x 106 cells were observed (Figure 3-3, right). In the second
set-up cultures with 2,3-dichloropyridine had initial cell numbers of 1 to 1.1 x 106 cells ml-1 and
cultures with 2,6-dichloropyridine had cell numbers of 0.9 to 1.2 cells ml-1. After 60 days of
incubation cell numbers ranged from 0.8 x 106 to 1 x 106 cells ml-1 for cultures with 2,3dichloropyridines or 2,6-dichloropyridines, respectively.
3.1.5
Brominated phenols as electron acceptor
Strain CBDB1 was cultivated with brominated phenols to investigate growth and the influence of
hydroxy substituents onto reductive debromination. For this, cultures with D. mccartyi strain CBDB1
and 2,4-dibromophenol, 2,6-dibromophenol or 2,4,6-tribomophenol were set up. First attempts to
cultivate strain CBDB1 with initial bromophenol concentrations of 50 µM were not successful.
Dehalogenation was slow and cell growth was low. Therefore, lower initial bromophenol
concentrations were tested. Successful cultivation of strain CBDB1 was achieved by applying initial
bromophenol concentrations of 10 µM and addition of further 20 µM of either 2,4-, 2,6- dibromo-, or
2,4,6-tribromophenol in repeated steps with continuing growth of strain CBDB1. D. mccartyi strain
CBDB1 was cultivated over 89 days with 2,4-dibromophenol, 2,6-dibromophenol and 2,4,6tribromophenol in separate cultures. First transformation products were detected after 7 days in
cultures with 2,4- and 2,6-dibromophenol and after 7 to 14 days in cultures with 2,4,6-tribromophenol.
Additional 20 µM of the respective bromophenols were added after 20 days, 40 days and 69 days to
cultures with strain CBDB1. In cultures with strain CBDB1 2,4-dibromophenol was transformed to
4-bromophenol and 2,6-dibromophenol to 2-bromophenol. Both monobromophenol congeners were
transformed subsequently to phenol (Figure 3-4, left). The first transformation product detected for
2,4,6-tribromophenol was 2,4-dibromophenol, which was transformed successively to 4-bromophenol
and phenol (Figure 3-4, left). Chemical controls contained 10 µM 2,4,6-tribromophenol,
2,4-dibromophenol or 2,6-dibromophenol. Additional 20 µM of the respective bromophenol were
added after 20 days and after 40 days of incubation. No reaction products were detected in chemical
controls with 2,4- or 2,6-dibromophenol. 2,4,6-Tribromophenol reacted in chemical controls
abiotically to 21.3 µM of 2,4-dibromophenol after 89 days of incubation. This corresponds to
approximately 50% of 2,4,6-tribromophenol transformed in cultures incubated with strain CBDB1. No
further abiotic transformation of 2,4-dibromophenol or monobromophenol was observed in the
chemical control.
40
Results
80
1.2e+7
1.0e+7
-1
cell growth (cells ml )
concentration (µM)
60
40
8.0e+6
6.0e+6
4.0e+6
20
2.0e+6
0
0.0
0
20
40
60
80
100
BPh ,6-DBPh ,6-TBPh
2
2,4-D
2,4
electron acceptor
time (days)
Figure 3-4: Transformation of brominated phenols and cell numbers of strain CBDB1. Left: Phenol production
in cultures of strain CBDB1 amended with 2,4-dibromophenol (○), 2,6-dibromophenol (▼) or 2,4,6tribromophenol (●). Right: Cell numbers of strain CBDB1 in cultures with one of the three tested bromophenols
after inoculation (black bars) and after 89 days of incubation (grey bars). Shown are means of triplicate cultures
± SD.
Cultures with 2,4-dibromophenol had initial cell numbers of 1 to 1.6 x 106 and final cell numbers
of 2.2 to 6.6 x 106 cells ml-1 (Figure 3-4, right). Growth yields of 0.3 x 1014 cells mol-1 halogen
released were calculated. Cultures with 2,6-dibromophenol had initial cell numbers of 1.5 to 2.1 x 106
cells ml-1 and final cell numbers of 6.8 to 7.2 x 106 cells ml-1 after 89 days of incubation (Figure 3-4,
right), corresponding to a growth yield of 0.37 x 1014 cells mol-1 of bromide released. Cultures with
2,4,6-tribromophenol had initial cell number of 1.6 to 2.3 x 106 cells ml-1. After 89 days of cultivation
0.5 to 1.1 x 107 cells ml-1 were obtained (Figure 3-4, right). After subtraction of the abiotic background
reaction of 2,4,6-tribromophenol measured in the chemical control, growth yields of 0.4 x 1014 cells
mol-1 halogen released were calculated.
3.1.6
Brominated furoic acids as electron acceptor
Strain CBDB1 was cultivated with brominated furoic acids to investigate growth with brominated
furoic acids and the influence of the oxygen heteroatom onto reductive debromination. The
transformation of 4,5-dibromo-2-furoic acid and 5-bromo-2-furoic acid was measured by the release
of bromide. First attempts to measure the release of bromide with an ion selective electrode were
hampered by the high chloride concentration in the medium. Therefore, bromide release was measured
by ion chromatography. Ion chromatography did not allow for the detection of the dehalogenated
reaction products. However, 5-bromo-2-furoic acid contains only one halogen substituent, therefore
dehalogenation will yield a non-halogenated reaction product. 4,5-Dibromo-2-furoic acid was not
stable under the chosen cultivation conditions and bromide was released also in the abiotic chemical
41
Results
controls. Therefore the release of bromide in the abiotic control was subtracted from the release of
bromide in cultures with strain CBDB1. In the first cultivation set-up with 4,5-dibromo-2-furoic acid,
the abiotic chemical control contained 164 µM of bromide after 24 hours of incubation. In cultures
with strain CBDB1 set up in triplicates, 100 µM to 172 µM of bromide were measured after 24 hours
of incubation. After 31 days of incubation 276 µM bromide were detected in the abiotic chemical
control. In cultures with strain CBDB1 503 ± 90 µM bromide were detected. A bromide release of 250
± 60 µM by microbial reductive dehalogenation was calculated after subtraction of the abiotic bromide
release in the chemical control and subtraction of the initial bromide concentration measured after 24
hours of incubation in cultures with strain CBDB1. In a second transfer, bromide concentrations after
addition of 4,5-dibromo-2-furoic acid and 24 h equilibration were 80 ± 17 µM. After 34 days of
incubation 140 ± 10 µM were measured in cultures with strain CBDB1, however in the chemical
control 130 µM of bromide were released abiotically. After 127 days, only two of three parallel
cultures showed a high bromide release of 740 and 1240 µM compared to the chemical control, in
which 590 µM of bromide were detected, corresponding to a release of 402 ± 252 µM bromide by
reductive dehalogenation (Figure 3-5, left).
In the first cultivation set-up with 5-bromo-2-furoic acid, bromide concentrations after 24 hours of
incubation were 85 to 116 µM in triplicate cultures. The abiotic chemical control contained 131 µM
bromide after 24 hours of incubation. After 31 days of incubation, 110 to 178 µM bromide were
detected in cultures with strain CBDB1. In abiotic chemical controls no abiotic reaction of 5-bromo-21e+8
1200
-1
cell growth (cells ml )
bromide concentration (µM)
1000
800
600
400
1e+7
200
0
1e+6
A
B-2-F
4,5-D
-2-FA
5-MB
electron acceptor
A
B-2-F
4,5-D
-2-FA
5-MB
electron acceptor
Figure 3-5: Transformation of brominated furoic acids and concomitant growth of strain CBDB1. Left: Bromide
release in cultures of strain CBDB1 amended with 4,5-dibromo-2-furoic acid or 5-bromo-2-furoic acid. The
initial bromide concentration measured in cultures with strain CBDB1 and the bromide release detected in the
abiotic chemical controls after 127 or 63 for 4,5-dibromo- or 5-bromo-2-furoic acid respectively, were subtracted
from the final bromide concentration measured in cultures with strain CBDB1. Right: Cell numbers of strain
CBDB1 in cultures with one of the brominated furoic acids after inoculation (black bars) and after 127 days (4,5dibromo-2-furoic acid) or 63 days (5-bromo-2-furoic acid) of incubation (grey bars). Data for 4,5-dibromo-2furoic acid show two parallel cultures ± SD and for 5-bromo-2-furoic acid three parallel cultures ± SD.
42
Results
furoic acid was observed and the bromide concentration remained at 131 µM. In a second transfer with
5-bromo-2-furoic acid, initial concentrations of 156 ± 19 µM of bromide were measured in cultures
and 165 µM bromide in the abiotic chemical control. After 63 days of incubation, 823 ± 300 µM
bromide were detected in cultures with strain CBDB1 and 108 µM bromide in the abiotic chemical
control, corresponding to a release of 666 ± 279 µM bromide (Figure 3-5, left).
Cell growth was detected with 4,5-dibromo-2-furoic acid and 5-bromo-2-furoic acid. In the first
set-up with 4,5-dibromo-2-furoic acid cells of the inoculum were previously cultivated on 1,2,3trichlorobenzene. Initial cell numbers were 1.7 to 2.4 x 106 cells ml-1 in three parallel cultures. The
final cell numbers after 31 days of incubation were 0.8 to 1.5 x 107 cells ml-1, corresponding to a cell
growth of 0.4 x 1014 cells mol-1 released. In the second transfer, initial cell numbers in cultures with
4,5-dibromo-2-furoic acid were 2.5 to 6.6 x 106 cells ml-1. In the two cultures with a higher bromide
release compared to the chemical control, cell numbers increased to 0.9 and 1.8 x 107 cells ml-1 after
127 days (Figure 3-5, right), corresponding to a growth yield of 0.1 x 1014 cells mol-1 halogen released.
In the first set-up of cultures with 5-bromo-2-furoic acid initial cell numbers ranged from 1.9 to 2.6 x
106 cell ml-1 and increased within 31 days to 0.2 to 1.3 x 107 cells ml-1, which corresponds to a growth
yield of 1 x 1014 cells mol-1 halogen released. In the second transfer, cell numbers ranged from 3.5 to
4.3 x 106 cells ml-1 and increased to 6.2 to 7.4 x 107 cells ml-1 within 63 days (Figure 3-5, right).
Growth yields of 0.95 x 1014 cells mol-1 halogen released were calculated.
3.1.7
Brominated benzoic acids as electron acceptor
The influence of methoxy and hydroxy substituents onto reductive debromination was
investigated with brominated hydroxy- and methoxybenzoic acids. Additionally, growth of strain
CBDB1 with brominated benzoic acids was investigated. The transformation of brominated benzoic
acids was monitored by measuring the bromide ion concentration in the culture medium via ion
chromatography. Because both tested brominated benzoic acids contained only one halogen
substituent the expected dehalogenation products were 3,5-dimethoxy- or 3,5-dihydroxybenzoic acid.
Cultures of strain CBDB1 amended with 4-bromo-3,5-dimethoxybenzoic acid crystals, had a
concentration of about 41 ± 11 µM bromide after 24 hours of incubation. In the chemical abiotic
control 48 µM bromide was measured after 24 hours of incubation. After 102 days of incubation 57
µM bromide were detected in the chemical abiotic control. In cultures with strain CBDB1, 141 ± 83
µM bromide was detected after 102 days of incubation, corresponding to a release of approximately
100 ± 93 µM bromide. In a second transfer, a concentration of 35 µM bromide after addition of 4bromo-3,5-dimethoxybenzoic acid crystals and 24 hours of incubation was measured in the abiotic
chemical control and 58 ± 15 µM in cultures with strain CBDB1. After 63 days of incubation 24 µM
were detected in the chemical abiotic control, while in cultures with strain CBDB1 364 ± 15 µM were
detected, corresponding to a release of 305 ± 133 µM bromide (Figure 3-6, left). 43
Results
In the first set-up of three parallel cultures with 4-bromo-3,5-dihydroxybenzoic acid initial
bromide concentrations in the medium after addition of crystals were 11 ± 0.2 µM in cultures with
strain CBDB1 and 14 µM in the abiotic chemical control. In the abiotic chemical control, the bromide
concentration did not increase after 63 days of incubation, however in cultures with strain CBDB1
bromide concentrations increased to 110 ± 77 µM after 73 days of incubation. In the second transfer,
only in two of three parallel cultures with strain CBDB1 and 4-bromo-3,5-dihydroxybenzoic acid, an
increase of the bromide concentration was detected. During 63 days of incubation, in one culture the
bromide concentration increased from 20 to 95 µM bromide, while in a second culture the bromide
concentration increased from 38 to 792 µM (Figure 3-6, left).
In the set-up of cultures with 4-bromo-3,5-dimethoxybenzoic acid with cells previously cultivated
on 1,2,3-trichlorbenzene initial cell numbers ranged from 2.5 to 3 x 106 cells ml-1. After 31 days no
cell growth was detected. After 102 days of incubation 0.8 to 4.2 x 107 cells ml-1 were observed and a
growth yield of 1.5 x 1014 cells mol-1 halogen released was calculated. In the second transfer with 4bromo-3,5-dimethoxybenzoic acid growth yields of 1.9 x 1014 cells mol-1 halogen were calculated, cell
numbers increased from 4.0 to 5.6 x 105 cells ml-1 to 4.3 to 6.3 x 107 cells ml-1 during 63 days of
incubation (Figure 3-6, right).
4-Bromo-3,5-dihydroxybenzoic acid cultures had initial cell numbers of 1.7 to 2.4 x 106 cells ml-1
and increased after 39 days to 4.1 to 8.1 x 106 cells ml-1 but decreased slightly after 73 days to 4.1 to
800
1e+9
600
1e+8
-1
cell growth (cells ml )
bromide concentration [µM]
6.3 x 106 cells ml-1. Growth yields in the first set-up were 0.3 x 1014 cells mol-1 halogen released. In the
400
1e+6
200
0
1e+7
1e+5
BA
BA
,5-DM 4-B-3,5-DH
4-B-3
BA
BA
,5-DM 4-B-3,5-DH
4-B-3
electron acceptor
electron acceptor
Figure 3-6: Transformation of brominated benzoic acids and concomitant growth of D. mccartyi strain CBDB1.
Left: Bromide concentration in cultures of strain CBDB1 amended with one of the brominated benzoic acids
after 63 days of incubation. The initial bromide concentration was subtracted from the bromide concentration
measured after 63 days. Right: Cell numbers of strain CBDB1 after inoculation (black bars) and after 63 days of
incubation (grey bars). For 4-bromo-3,5-dimethoxybenzoic acid data show the mean of three parallel cultures ±
SD while for 4-bromo-3,5-dihydroxybenzoic acid data show the mean of two parallel cultures ± SD.
44
Results
second transfer, the initial cell number was 1 x 106 ± 1.63 x 104 cells ml-1 in three parallel cultures.
Only in the two cultures with increased bromide concentrations, cell numbers increased after 63 days
of incubation. Cell numbers ranging from 1 to 2.8 x 107 cells ml-1 (Figure 3-6, right). Calculated
growth yields from the two active cultures were 0.5 x 1014 cells mol-1 halogen released.
3.2
Set-up of a photometric microtiter plate-based resting cell activity assay
A photometric microtiter plate-based activity assay was established to allow for a fast screening
and identification of new electron acceptors of organohalide-respiring Dehalococcoidia and
microorganisms independent of analytical methods, which require establishment of specific programs
for the detection of individual dehalogenation products. D. mccartyi strain CBDB1 was used for this
purpose as a model organism.
3.2.1
Material choice for a 96-well microtiter plate for dehalogenase activity measurement
The photometric assay was initially set up in 96-well microtiter polystyrene plates with 200 µl
activity assay reaction mix (see also 2.5.1) and 20 or 40 µl of whole resting cell suspension of strain
CBDB1 previously concentrated with a rotary evaporator or a filter (see also section 2.4.1). The
oxidation of methyl viologen was monitored with a microtiter plate reader at 578 nm for 15 hours after
sealing the plate with a transparent plastic film (see also 2.5.3). Additionally the absorbance was
measured at 450 nm to correct for the absorbance by condensed water on the sealing plastic film. The
negative control contained strain CBDB1 resting cells in cultivation medium and 1,3-dichlorobenzene
in the activity assay reaction mix. 1,3-Dichlorobenzene was shown previously not to be transformed
by strain CBDB1 (7). The positive control contained strain CBDB1 resting cells in cultivation medium
and 1,2,3-trichlorobenzene dissolved in acetone as electron acceptor. Oxidation of methyl viologen in
the positive control was higher than in the negative control during the first 100 minutes of measuring
time. After approximately 100 minutes however, the oxidation of methyl viologen in the positive
control was similar to the oxidation measured in the negative control. GC-FID-based analysis of the
activity assay reaction mix containing 1,2,3-trichlorobenzene incubated in wells of the polystyrene
microtiter plate without resting cells, revealed a very low electron acceptors concentration in the
reaction mix after only one hour of incubation (6 ± 0.4 µM 1,2,3-trichlorobenzene). The very low
electron acceptor concentration could explain similar methyl viologen oxidation rates in the positive
control as in the negative control after approximately 100 minutes of incubation. Additionally,
measured oxidation rates showed high deviations between different wells within the same
experimental set-up. The polystyrene plate contained raised rims which did not allow for uniform
sealing of all wells by the plastic film allowing for oxygen penetration from the gas phase of the
anoxic glove box into wells. This could explain deviations between different rates measured for the
same reaction set-ups.
45
Results
To assure sufficient concentration of electron acceptors during fifteen hours of incubation and
uniform sealing of the wells, the activity assay was established in a 96-well microtiter glass plate to
minimize hydrophobic interactions between the halogenated compounds and the microtiter plate
material. Additionally, a tenfold higher electron acceptor concentration (625 µM) was applied to the
reaction mix. The assay was set up similar to the polystyrene plate with 200 µl reaction mix and 20 or
40 µl whole resting cell suspensions per well. The positive control contained strain CBDB1 resting
cells and 1,2,3-trichlorobenzene dissolved in acetone as electron acceptor. The negative control
contained strain CBDB1 cells and acetone but no electron acceptor. To test for abiotic reactions of the
investigated halogenated compounds, chemical controls were set up with each halogenated compound,
the activity assay reaction mix but no cells. Furthermore, controls were included containing only the
reaction mx without halogenated electron acceptor or cell suspension. The absorbance was measured
every 7.5 minutes for at least 700 minutes at 578 nm and 450 nm. In the chemical control without cells
the absorbance at 578 nm decreased only slightly from 1.05 to 0.99 within 700 minutes of incubation
(Figure 3-7). In the negative control, the absorbance increased from 1.05 to 1.1 within the first 52
minutes of incubation due to the reaction of methyl viologen with additionally introduced
titanium(III)citrate with the resting cell inoculum and then decreased continuously to 0.29. This
demonstrated that strain CBDB1 resting cells alone oxidized methyl viologen in the absence of an
electron acceptor. In the positive control the absorbance decreased with a faster initial rate from 1.06
to 0.1 (Figure 3-7). The activity was calculated in nmol s-1 ml-1 from the linear part of the slope as
indicated in Figure 3-7, when background activity was not subtracted. After subtraction of the
background oxidation of methyl viologen the activity was calculated in nkat ml-1 or nkat mg-1 whole
1.2
absorbance (578nm)
1.0
w/o cells, w/o e acceptor
0.8
cells, w/o e acceptor
0.6
0.4
0.2
cells and e- acceptor
0.0
0
200
400
600
time (min)
Figure 3-7: Resting cell activity measured at 587 nm in a glass microtiter plate. Methyl viologen oxidation in the
chemical control containing the reaction mix, medium without cells and no electron acceptor ( ). Methyl
viologen oxidation in the negative control containing the reaction mix, resting strain CBDB1 cells in cultivation
medium and acetone ( ). Methyl viologen oxidation in the positive control containing the reaction mix, strain
CBDB1 cells in cultivation medium and 1,2,3-trichlorobenzene from an acetonic stock solution as electron
acceptor ( ). The dashed lines indicate the part of the slope which was used for the calculation of strain CBDB1
resting cell activity.
46
Results
cell protein (see also for more information about background activity the following chapter 3.2.2)
using the formula described in Figure 2-4.
3.2.2
Analysis of background oxidation of methyl viologen in the microtiter plate activity
assay
The photometric activity assay is based on the measurement of the oxidation of methyl viologen
absorbance at 578 nm, reflecting the catalytic activity of reductive dehalogenases transferring
electrons from the reduced methyl viologen onto the halogenated compound (Figure 2-2). However,
the photometric measurement revealed the oxidation of methyl viologen with resting cells even
without halogenated electron acceptor. Potential reasons for this background oxidation were a)
halogenated electron acceptors introduced together with the resting cells, b) cultivation medium
components, introduced with the resting cells, c) reductive dehalogenases reducing a so far unknown
none-halogenated electron acceptor and d) living or dead parts of resting strain CBDB1 cells
catalysing the transfer of electrons onto a non-halogenated electron acceptor or parts of the cell acting
as electron acceptor.
3.2.2.1
Analysis of a potential electron acceptor carry-over with the inoculum
To investigate whether halogenated electron acceptors from the cultivation medium were carriedover to the activity test via the cell preparation, resting cell suspensions of strain CBDB1 were
prepared as described for the activity assay, extracted with hexane and analysed by GC-FID. Resting
cell suspension originating from cultures grown with 2-chloro-6-fluorobenzonitrile 1,2,3trichlorobenzene or 2,3-dichloroanilines were analysed but none of the electron acceptors were
detected as traces in the cell suspensions.
3.2.2.2 Medium components and vitamins show low influence on background activity
The influence of medium components on the background activity was tested by comparing the
oxidation of methly viologen in resting cell activity assays with i) strain CBDB1 cells in medium with
vitamins (see also 2.3.1.3 for detailed descrition of vitamins in the medium), ii) a control with
cultivation medium containing vitamins but no cells and iii) a second control with medium prepared
without vitamins and without cells. For each set-up the oxidation of methyl viologen was compared
between samples containing 1,2,3-trichlorobenzene as halogenated electron acceptor with samples
containing acetone and no halogenated electron acceptor. Acetone was chosen because 1,2,3trichlorobenzene was added to the activity assay reaction mix from an acetonic stock solution, which
can introduce oxygen into the test and therefore contribute to methyl viologen oxidation. For a better
comparability, the absorbance was calculated as oxidation of methylviologen in nmol s-1 ml-1 from the
linear part of the slope (Figure 3-8, A).
47
Results
In samples with strain CBDB1 resting cells and 1,2,3-trichlorobenzene the methyl viologen
oxidation was about twice as high as it was in samples with resting cells and acetone (Figure 3-8, A).
In samples without cells but with medium and vitamins, the methyl viologen oxidation was the same
in the set-up with 1,2,3-trichlorobenzene or without. Both corresponded to about one third of the
methyl viologen oxidation detected in samples with cells but without 1,2,3-trichlorobenzene,
demonstrating that resting cells account for about two third of the detected background methyl
viologen oxidation. In the experimental set-up with medium, without vitamins (see for medium
components section 2.3.1) and without cells, a similar oxidation of methyl viologen was detected for
the set-up with 1,2,3-trichlorobenzene as for the set-up with vitamins, medium but without cells
(Figure 3-8, A). This suggests that vitamins such as B12 were not involved in abiotic methyl viologen
oxidation and 1,2,3-trichlorobenzene reduction. However, even lower methyl viologen oxidation was
detected in the set-up with activity assay reaction mix medium without vitamins and without 1,2,3trichlorobenzene. Therefore, in chemical controls with the halogenated compounds but without resting
cell suspension were included inot activity assays.
3.2.2.3 Analysis of the influence of reductive dehalogenase activity on the background
methyl viologen oxidation
Previous experiment showed that the resting cell inoculum itself contributed to background
oxidation of methyl viologen in the microtiter plate in absence of a halogenated electron acceptor. To
investigate whether reductive dehalogeanses are involved in the backgound oxidation of methyl
viologen by tranferring electrons onto a non-halogenated, so far not identified electron acceptor,
reductive dehalogenase inhibition studies were conducted. For this strain CBDB1 cells were treated
with 1 mM propyl iodide as described in section 2.5.5. The inhibtion of reductive dehalogenation was
tested in the microtiter plate assay and in a parallel GC-FID-evaluated assay. The GC-FID-evaluated
assay was done to prove light reversibility of the inhibition. The production of 1,3-dichlorobenzene
from 1,2,3-trichlorobenzene was measured after 5 hours and activities in nmol s-1 ml-1 were calculated.
Samples containing 1,2,3-trichlorobenzene and propyl iodide showed 28% (~12 µM 1,3dichlorobenzene produced) of the activity compared to samples with 1,2,3-trichlorobenzene without
propyl iodide ( ~45 µM 1,3-dichlorobenzene produced). The activity was restored to about 50% of the
original activity after exposure to light (~22 µM 1,3-dichlorobenzene produced). In parallel, cells
treated the same way were applied to the microtiter plate activity assay. While the activity of strain
CBDB1 resting cells with 1,2,3,4-tetrachlorobenzene was reduced to about 82% of the original
activity, the activity of strain CBDB1 resting cells with 1,2,3-trichlorobenzene was reduced to about
68% of the original activity. In contrast, the background oxidation of methyl viologen in negativ
controls without halogenated electron acceptor was not reduced by propyl iodide compared to the
control without propyl iodid, indicating that the corrinoid-containing reductive dehalogenases did not
participate in the background methyl viologen oxidation (Figure 3-8, B). These results were supported
48
Results
by experiments with ethyl iodide as reductive dehalogenase inhibitor. Samples with 1 mM ethyl iodide
showed approximately 50% of the original activity with 1,2,3,4-tetrachlorobenzene and about 80% of
the original activity with 1,2,3-trichlorobenzene in the microtiter plate activity assay, however, the
background methyl viologen oxidation did not decrease after ethyl iodide treatment.
3.2.2.4
Heat treated cells show decreased background methyl viologen oxidation
By incubating the cells at different temperatures before using them for the activity assay it was
shown that heat-labile components (e.g. proteins) within the cells are responsible for the background
activity. The background activity was around 0.01 nmol s-1 ml-1 in tests with intact cells but without
electron acceptor (Figure 3-8, C, grey bar at 25°C) and a strong activity of reductive dehalogenases
was measured when the cells were incubated with 1,2,3-trichlorobenzene as electron acceptor (black
bar, 25°C). The reductive dehalogenase activity was destroyed by preincubation at 60°C so that the
activity with electron acceptor was the same as the activity without electron acceptor. By
preincubating the cells at 80°C also the background activity dropped by about 90% with or without
electron acceptor, indicating, the components responsible for the background activity were inactivated
at 80°C.
3.2.2.5 Analysis of methyl viologen oxidation in the presence of bacteria with organohalide
respiration-independent mode of living
Because the previous tests showed that bacterial cell mass was responsible for the background
activity, it was also tested if this was a specific effect of cells of strain CBDB1 or if other cells types
which are not depending on organohalide respiration induce the background oxidation of methyl
viologen. This was tested by adding E. coli cells to the activity test and comparing obtained methyl
viologen oxidation rates with the ones obtained from samples with strain CBDB1. E .coli K12 strain
was grown on LB-medium, harvested and diluted in fresh sterile medium otherwise used for
cultivation of strain CBDB1 to obtain a final cell number of 1.6 x 107 cells ml-1 corresponding to cell
numbers of the strain CBDB1 inoculum for activity assays. The E. coli K12 sample was then used as
an inoculum for the activity assay and measured with 1,2,3-trichlorobenzene and without electron
acceptor but acetone. The methyl viologen oxidation in samples with strain CBDB1 but without
electron acceptor amounted to about half of the methyl viologen oxidation observed for samples with
1,2,3-trichlorobenzene (Figure 3-8, D). E. coli cells induced an oxidation of methyl viologen
corresponding to about half of the background methyl viologen oxidation induced by strain CBDB1
(Figure 3-8, D). Together, results suggested that reductive dehalogenases were not involved in the
catalysis of background oxidation of methyl viologen but that another heat-labile factor in strain
CBDB1 was responsible for the background activity. For all further calculations of activity, the
background oxidation of methyl viologen in negative controls with cells but without halogenated
49
Results
electron acceptor were subtracted from the total methyl viologen oxidation in samples with acceptor
0.030
1,2,3-TCB
w/o electron acceptor
A)
-1 -1
methyl viologen oxidation (nmol s ml )
methyl viologen oxidation (nmol s-1 ml-1)
but acetone.
0.025
0.020
0.015
0.010
0.005
0.000
it
it
ed
with v m w/o v
1 in m
CBDB medium mediu
0.16
w/o propyl iodide
with propyl iodide
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
r
B
B
-TeC 1,2,3-TC e- accepto
1,2,3,4
w/o
1,2,3-TCB
w/o electron acceptor
electron acceptor
C)
-1 -1
methyl viologen oxidation (nmol s ml )
-1 -1
methyl viologen oxidation (nmol s ml )
medium components
0.04
0.03
0.02
0.01
0.05
1,2,3-TCB
w/o electron acceptor
60°C
80°C
0.03
0.02
0.01
c
D. mc
temperature
D)
0.04
0.00
0.00
25°C
B)
artyi
1
CBDB
K12
E.coli
strain
Figure 3-8: Investigation of parameters influencing the background oxidation of methyl viologen (MV) in the
photometer-based 96-well microtiter plate activity assay. A) Influence of vitamins and medium components
onto MV oxidation. Compared were resting CBDB1 cells with medium and vitamins, medium with vitamins
without resting cells and medium without resting cells and without vitamins B) Oxidation of MV in positive
controls with cells but without inhibitor and with cells and the corrinoid inhibitor propyl iodide. Shown are
results for 1,2,3,4-TeCB, 1,2,3-TCB or without halogenated electron acceptor. C) MV oxidation detected with
strain CBDB1 cells exposed to different temperatures in controls with 1,2,3-TCB and in negative controls
without electron acceptor D) Oxidation of MV induced by E. coli K12 cells compared to strain CBDB1 cells in
presence of 1,2,3-TCB and in absence of a halogenated electron acceptor. Abbreviations: MV = methyl
viologen, med = medium, vit = vitamins, w/o = without, e- acceptor = electron acceptor.
50
Results
3.2.1
A total number of 560 cells are required for activity detection in 96-well microtiter
plate tests
The photometric activity assay was established to screen for electron acceptors of pure
dehalogenating cultures, dehalogenating enrichment cultures, microcosms or cells extracted from
sediments. Therefore, the minimum cell number required for detection of activity in the photometric
assay was investigated using an actively dehalogenating culture of Dehalococcoides mccartyi strain
CBDB1 which was grown to 6.2 x 106 cells ml-1. Cells were concentrated using a 0.2 µm filter (see
section 2.4.2, page 19) to cell numbers of 1.4 x 107 cells ml-1. Additionally, a D. mccartyi strain
CBDB1 culture was tested, which was initially grown on 1,2,3-trichlorobenzene to reach cell numbers
of approximately 1.7 x 107 cells ml-1 but was subsequently not amended with additional doses of 1,2,3trichlorobenzene. The culture was six months old when used for the assay. Cell numbers after
harvesting of cells from the medium were 1.1 x 107 cells ml-1. The starvation conditions were
investigated to test for the limits of the microtiter plate activity assay to detect organohalide-respiring
Dehalococcoidia from marine sediments, which possibly reside under starvation conditions in marine
sediments. Cell numbers of 1.4 x 107, 2.8 x 106, 1.4 x 106, 2.8 x 105, 1.4 x 105 and 2.8 x 104 cells ml-1
were tested. Activity with 1,2,3-trichlorobenzene was still detected when using an inoculum of 20 µl
with 2.8 x 104 cells ml-1 from the actively dehalogenating culture, corresponding to 560 cells per well
in 220 µl activity assay reaction mix. For cells from the starvation culture of strain CBDB1, activity
was detected at 2.8 x 105 cell ml-1 corresponding to 5600 cells per well in 220 µl reaction mix. With
cell numbers of 1.4 x 105 or 2.8 x 104 cells ml-1 it was not possible to detect activity with this culture,
indicating that the age and growth phase of strain CBDB1 cultures contributes to the activity rates
measured in activity assays.
3.2.2
Microtiter plate-analysed activities are comparable to GC-FID-analysed activities
Parallel to each photometric measurement, resting cell suspensions of strain CBDB1 were used to
set up activity assays in HPLC vials for further analysis by GC-FID as described previously (95, 190).
The activities analysed by GC-FID were compared with activities analysed in parallel by the
photometer in 96-well microtiter plates. For this, cells were cultivated with 1,2,4-tribromobenzene,
harvested and concentrated by rotary evaporation. Subsequently cells were either applied to the
microtiter plate activity assay or incubated for 4 h in 1.5 ml HPLC vials with the reaction mix
containing either 1,2,3-trichlorobenzene or one of several brominated benzenes. GC-FID analysis of
the reaction products showed 1,2,3-trichlorobenzene was dehalogenated to 20.3 µM 1,3dichlorobenzene within 240 min, corresponding to an activity of 0.016 nkat ml-1 cell suspension
compared to 0.017 nkat ml-1 cell suspension measured in the photometric assay. For brominated
benzenes the lowest activity in the GC-based assay was measured for 1,4-dibromobenzene with ~0.01
nkat ml-1 and highest activities for 1,2,4-tribromo- and 1,2-dibromobenzene with 0.03 nkat ml-1. In the
51
Results
photometric assay activities ranged from 0.01 for 1,4-dibromobenzene and 0.04 nkat ml-1 for 1,2,4tribromobenzene. The reaction products of 1,2,4-tribromobenzene detected in the GC-based resting
cell activity assay were mainly 1,4-dibromobenzene, in some samples within the defined incubation
time a small amount of monobromobenzene was formed. The first dehalogenation product of the three
tested dibromobenzenes was monobromobenzene, benzene was not observed. Dehalogenation of
monobromobenzene was neither detected in the photometric assay nor in the GC-based assay. When
activities were calculated using a value of 30 fg of protein per single cell of strain CBDB1 (personal
communication, Anja Kublik, UFZ, Leipzig) activities ranging from 8–38 nkat mg-1 protein for the
photometric assay and 7–30 nkat mg-1 protein for the GC-based assay were obtained (Table 3-1).
Table 3-1: Specific activities in nkat mg protein-1 obtained in photometer or GC-based activity assays when
assuming 30 fg protein per cell of strain CBDB1. Strain CBDB1 was cultivated previously with 1,2,4tribromobenzene.
electron-acceptor for
the assay
MBB a
1,2-DBB a
1,3-DBB a
1,4-DBB a
1,2,4-TBB a
1,2,3-TCB a
3.3
Photometric assay
Specific activity
Gas chromatographic assay
Specific activity
Dehalogenation products
0
35.5 ± 2
16 ± 1
8 ± 2.5
37.7 ± 6.2
16.9 ± 0.9
0
29.8
21.0
7.3
28.3
15.8
none
MBB
MBB
MBB
1,3-DBB
1,3-DCB
Analysis of enzyme activity of D. mccartyi strain CBDB1
After establishing the microtiter plate resting cell activity assay, the assay was used to investigate
activity rates of strain CBDB1 with different halogenated and non-halogenated compounds.
Additionally, the influence of the electron acceptor used for cultivation onto activities measured in the
resting cell assays was investigated.
3.3.1
Specific activities of resting cells of D. mccartyi strain CBDB1 with halogenated
compounds
Cells of strain CBDB1 used as an inoculum for the activity assay were grown with 1,2,3trichlorobenzene or hexachlorobenzene as electron acceptor. Thus, dehalogenation activity was based
on reductive dehalogenases expressed during 1,2,3-trichlorobenzene or hexachlorobenzene cultivation.
Highest specific activities were measured for 2,4,6-tribromophenol (87.4 nkat mg-1), 2,3dichlorobenzonitrile (50.6 nkat mg-1) and 2,3-dichlorothiophene (42.1 nkat mg-1) (Table 3-2). The
lowest activity was detected for 2,5-dichlorothiophene (4.5 nkat mg-1) (Table 3-2). No activity was
measured for 2,3-dichloroaniline, 2,5-, 2,6-dichlorobenzonitrile, 2-chloro-6-fluorobenzonitrile, 4bromo-3,5-dihydroxybenzoic acid and 5-bromo-2-furoic acid although dehalogenation and growth was
observed for all six electron acceptors during cultivation with the same substrates (Table 3-2). For 4,5-
52
Results
dibromo-2-furoic acid a strong abiotic oxidation of methyl viologen was detected in the chemical
control. Therefore, no activity data were determined for 4,5-dibromo-2-furoic acid. Resting cell
activity with 2-chloro-6-fluorobenzonitrile, 2,3-dichloroaniline, 4-bromo-3,5-dihydroxybenzoic acid
or monobromobenzene was not observed despite the transformation of the respective compounds in
cultivation (Table 3-2).
Table 3-2: Strain CBDB1 resting cell activity. Specific activities were calculated using 30 fg per cell of strain
CBDB1.
Electron acceptor
Sp. act. [nkat mg-1] Cultivation e- acceptor
2,3-DCA
2,4-DCA
2,6-DCA
2,3-DCBN
2,5-DCBN
2,6-DCBN
2-C-6-FBN
2,4,6-TBPh
2,4,6-TBPh
2,4-DBPh
2,4-DBPh
2,6-DBPh
2,6-DBPh
4-BPh
4-B-3,5-DMBA
4-B-3,5-DHBA
2,3-DCTh
2,5-DCTh
2-CTh
4,5-DB-2-FA
5-B-2-FA
2,3-DCPy
2,6-DCPy
3-B-2-CPy
PeCPh
2,3,4,5-TeCPh
2,4,6-TCPh
2,4-DCPh
2,6-DCPh
2,3-DCPh
4-BAn
4-BIn
Non-halogenated eacceptor
humic acids (Sigma
Aldrich)
anthraquinone
2,6-DM-1,4-BQ
hydroquinone
0
0
0
50.6 ± 4.5
0
6±8
0
87.4 ± 3.7
27.1 ± 0.5
41.7 ± 3.9
18.1 ± 0.5
14.3 ± 1.9
24.2 ± 1.4
1.0 ± 0.2
34.0 ± 4.7
0
42.1 ± 1.3
4.5 ± 1.1
3.6 ± 0.2
Abiotic reaction
0
0
0
30.2 ± 1.7
21.4 ± 0.8
25.0 ± 0.7
1.2 ± 0.3
1.7 ± 0.6
1.9 ± 0.7
2.5 ± 0.3
0
0
1,2,3-TCB/2,3-DCA
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB/2-C-6-FBN
1,2,3-TCB
HCB
1,2,3-TCB
HCB
1,2,3-TCB
HCB
HCB
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
HCB
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
HCB
HCB
HCB
HCB
HCB
HCB
1,2,3-TCB
1,2,3-TCB
Activity in
cultivation
Active
Not active
Not active
Active (199)
Active (199)
Active (199)
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active (234)
Active (234)
Active (234)
Active
Active
n.a.d.
n.a.d.
Active (234)
Active (119)
Active (119)
Active (119)
Active (119)
Active (119)
Active (119)
n.a.
n.a.
0
1,2,3-TCB
n.a.
0
0
0
1,2,3-TCB
1,2,3-TCB
1,2,3-TCB
n.a.
n.a.
n.a.
Abbreviations: n.a.: not analysed; n.a.d. no activity detected.
53
Results
3.3.2
Attempts to improve sensitivity of the activity assay
To test whether an artificial electron donor with a more negative redox potential could improve
resting cell activity in the microtiter plate assay, the activity assay was set up with ethyl viologen as
artificial electron donor. Ethyl viologen has a redox potential of -480 mV compared to methyl
viologen with -440 mV (190). However, ethyl viologen did also not lead to detectable reduction of
2,3-dichloroaniline, 2-chloro-6-fluorobenzonitrile or monobromobenzene in the activity assay. In
addition, activity assays were conducted in HPLC reaction vials which allowed for the extraction of
the activity assay reaction mix after incubation and a detection of reaction products by gas
chromatography. Resting cells of strain CBDB1 were incubated for up to 60 hours at 30°C with
activity
assay
reaction
mix
and
2-chloro-6-fluorobenzonitrile,
2,3-dichloroaniline
or
monobromobenzene. No transformation of monobromobenzene, 2-chloro-6-fluorobenzonitrile or 2,3dichloroaniline was observed when using gas chromatography for the analysis of reaction products,
even after the incubation of cells with the electron acceptor and activity assay reaction mix up to 60 h
until extraction.
3.3.3
Analysis of the influence of the electron acceptor used for cultivation onto resting
cell activity and reductive dehalogenase expression using brominated benzenes
As shown above, resting cells of strain CBDB1 grown with 1,2,3-trichlorobenzene showed
activity with diverse halogenated electron acceptors (Table 3-2). To investigate the influence of the
electron acceptor used for cultivation onto activity rates measured in the in vitro activity assay in more
detail, strain CBDB1 was cultivated with brominated and chlorinated benzenes and activity assays
were conducted and obtained rates were compared. Additionally, expression studies were conducted
using shotgun proteomics to investigate the induction of specific reductive dehalogenases with
different bromobenzene congeners. Brominated benzenes were used in the current study as model
compound because brominated compounds have been abundantly detected in marine environments
(139) and have relevance by resembling basic structures of brominated flame retardants (66, 191).
Additionally, they have the advantage that they do not contain other functional groups and substituents
which may introduce another level of complexity due to toxic or inhibitory effects such as for instance
observed for compounds with hydroxy substituents (see also 3.1.5).
3.3.3.1 Cross cultivation and activity assays with different brominated benzenes and
1,2,3-trichlorobenzene
Resting cells of D. mccartyi strain CBDB1 were tested in in resting cell activity assays with
monobromobenzene, 1,2-, 1,3-, 1,4-dibromobenzene and 1,2,4-tribromobenzene after their cultivation
with either 1,2,3-trichlorobenzene, monobromobenzene, 1,2-, 1,3-, 1,4-dibromobenzene or
1,2,4-tribromobenzene. All tests were done in triplicates using resting cells from three separate
54
Results
Table 3-3: Specific activities in nkat mg-1 whole cell protein of strain CBDB1 cultivated with different electron
acceptors and tested with different electron acceptors in the in vitro activity test. All numbers represent means of
three cultures tested in three microtiter plate assays with three replica wells each ± standard deviation.
e- acceptor
cultivation
1,2,3-TCB
MBB
1,2-DBB
1,3-DBB
1,4-DBB
1,2,4-TBB
1,2,3-TCB
36.6 ± 9.1
21.6 ± 8.7
16.5 ± 9.4
18.5 ± 9.2
17.0 ± 2.0
13.4 ± 7.8
MBB
-5.7 ± 4.7
-5.4 ± 4.9
0.0 ± 0.8
0.0 ± 0.6
2.9 ± 3.1
1.4 ± 1.9
e- acceptor in the activity assay
1,2-DBB
1,3-DBB
241 ± 21.5
11.4 ± 8.1
26.1 ± 14.4
7.1 ± 4.0
23.4 ± 16.5
11.6 ± 7.6
28.5 ± 14.6
11.2 ± 5.0
13.5 ± 4.7
8.4 ± 1.4
20.9 ± 17.3
10.9 ± 8.1
1,4-DBB
4.5 ± 1.4
1.5 ± 4.3
7.9 ± 3.5
7.3 ± 3.8
4.9 ± 2.8
7.1 ± 4.6
1,2,4-TBB
219 ± 110
28.4 ± 3.1
22.3 ± 11.9
31.7 ± 9.9
19.3 ± 1.4
25.8 ± 15.4
Abbreviations: TCB – trichlorobenzene, MBB – monobromobenzene, DBB – dibromobenzene, TBB –
tribromobenzene
cultures, cultivated with the same electron acceptor. In addition each inoculum of strain CBDB1 was
tested in triplicates with each electron acceptor in the 96-well microtiter plate activity assays (Table
3-3).
When comparing specific activities of 1,2,3-trichlorobenzene, monobromobenzene, 1,2-, 1,3-,
1,4-dibromobenzene or 1,2,4-tribromobenzene no enhanced specific activities were observed for a
specific electron acceptor after the cultivation of the strain CBDB1 inoculum with the same electron
acceptor except for 1,2,3-trichlorobenzene (Table 3-3 and Figure 3-9). Cells cultivated with
1,2,3-trichlorobenzene gave even higher activities for 1,2-dibromo- and 1,2,4-tribromobenzene than
for
1,2,3-trichlorbenzene.
Higher
specific
activities
with
1,2-dibromobenzene
or
1,2,4-tribromobenzene than with 1,2,3-trichlorobenzene were observed for all cultivation conditions
1000
e- acceptor = 1,2,3-TCB
e- acceptor for cultivation = e- acceptor for activity assay
-1
specific activity (nkat mg protein)
tested (Table 3-3). In addition, halogenated benzenes with singly flanked halogen substituents such as
100
10
1
1,2,3-TCB
MBB
1,2-DBB
1,3-DBB
1,4-DBB
1,2,4-TBB
electron acceptor for activity assay
Figure 3-9: Specific activities measured in resting cell activity assays. Resting cells were cultivated prior to
activity assays with 1,2,3-trichlorobenzene (black bars) or with the same electron acceptor which was used in the
activity assay (grey bars). Shown are the means of three separate 96-well microtiter plate photometer-based
activity assay measurements ± SD. Each of the replicas contained a different inoculum of strain CBDB1 grown
on the respective electron acceptor. TCB – trichlorobenzene, MBB – monobromobenzene, DBB –
dibromobenzene, TBB – tribromobenzene.
55
Results
1,2,4-tribromobenzene, 1,2-dibromobenzene or 1,2,3-trichlorobenzene gave higher specific activities
than halogenated benzenes with isolated halogen substituents, independently from the electron
acceptor used for cultivation. For bromobenzenes with isolated bromine substituents secondary
bromine substituents in meta-position enhanced activity compared to secondary bromine substituents
in para-position, leading to higher activities with 1,3-dibromobenzene than with 1,4-dibromobenzene
(Table 3-3). It was concluded, that chemical properties influenced the activity of resting cells of strain
CBDB1 in activity assays with different brominated benzenes more than the electron acceptor used for
cultivation. Additionally, it was shown that cultivation on bromobenzenes did not increase activity in
the resting cell assays with bromobenzenes. Instead, even higher activities were obtained when cells
were previously cultivated with 1,2,3-trichlorobenzene.
3.3.3.2 Expression of reductive dehalogenase after cultivation with brominated benzenes
Specific activities obtained in resting cell assays suggested the same reductive dehalogenases are
involved in dehalogenating of bromobenzene congeners or 1,2,3-trichlorobenzene. The expression of
reductive dehalogenases in D. mccartyi strain CBDB1 cultivated with monobromobenzene, 1,2- 1,3or 1,4-dibromobenzene or 1,2,4-tribromobenzene was therefore further analysed by shotgun
proteomics. In total, seven different reductive dehalogenases were identified in the different cultures.
The protein with the highest estimated protein abundance was CbdbA80 when using the emPAI-value
(exponentially modified protein abundance index) for an estimation of the relative abundance of
reductive dehalogenase proteins (172). CbdbA80 was previously reported to be expressed during
cultivation of strain CBDB1 with 1,2,4-trichlorbenzene (104). CbdbA80 was detected in all samples.
The 1,2,3-trichlorobenzene dehalogenating protein CbdbA84 (CbrA) (104) was detected only in
samples grown with 1,3-dibromobenzene, 1,4-dibromobenzene and 1,2,4-tribromobenzenes but not
with 1,2-dibromobenzene or monobromobenzene. CbdbA1455 was expressed in all samples and was
according to the emPAI value expressed at the same or at higher levels than CbdbA84.
Table 3-4: Expression of reductive dehalogenases after the cultivation with different bromobenzenes as electron
acceptors.
e--acceptor
RdhCBDB1
CbdbA80
CbdbA84
CbdbA88
CbdbA1453
CbdbA1455
CbdbA1618
CbdbA1638
MBB
1,2-DBB
1,3-DBB
1,4-DBB
1,2,4-TBB
Score
emPAI
Score
emPAI
Score
emPAI
Score
emPAI
Score
emPAI
221
0.75
614
2.44
461
306
1.60
0.51
1147
127
3.82
0.34
523
379
1.75
0.60
61
128
121
0.25
0.13
0.19
387
0.52
279
0.44
85
0.19
314
0.60
254
0.51
293
0.60
151
0.21
195
0.28
107
0.13
111
0.13
92
0.20
208
0.43
The score describes the Mascot protein score. It represents a probability value for the identification of a protein;
higher protein scores correspond to a more confident match between the experimental detection of ion m/z
values and expected m/z values for the respective protein; emPAI – exponentially modified protein abundance
index. A higher emPAI value indicates a higher abundance of an Rdh protein. Protein score and emPAI were
calculated from ion scores that exceeded the significance threshold (see material and methods).
56
Results
3.4
Potential for organohalide respiration in deep marine non-contaminated
sediments
Diverse reductive dehalogenase homologues have been detected in marine sediments (60, 61),
however little is known about the type and spectrum of electron acceptors transformed in marine
sediments. Marine microorganisms exposed to natural halogenated compounds could transform a wide
range of halogenated compounds from natural and anthropogenic sources and therefore play a
significant role for fate prediction of halogenated compounds in the environment.
In this part of the study it was investigated whether dehalogenation activity can be detected in
marine sediments from non-contaminated sites using the methyl viologen-based resting cell activity
assay, marine sediment microcosms and crude extracts or whole sediment slurries from marine noncontaminated sediment sites. The microcosms used in this study were set up in a parallel project by
Camelia Algora (UFZ Leipzig) with non-contaminated sediments from the Chilean continental margin
of 4.37–4.42 mbsf (192) using 1,2,3-trichlorobenzene. The microcosms were used to test for the
applicability of the microtiter plate resting cell activity assay to identify new electron acceptors of so
far uncharacterized dehalogenating microorganisms. Additionally, the dehalogenation activity was
further characterized for potentially involved co-factors, oxygen and temperature sensitivity.
Microcosms were tested for the presence of strain CBDB1 by PCR to assure that no crosscontamination between the cultures occurred (see also Table 2-12 and Table 2-17). No contamination
of strain CBDB1 was detected. In addition, crude sediments and sediment extracts were tested for
dehalogenation activity without previous enrichment procedures by cultivation.
3.4.1
Dehalogenation activity with brominated electron acceptors with cells of marine
microcosms from the coast off Chile
The potential of marine microcosms from sediments of the coast off Chile to transform
brominated compounds in spite of their previous enrichment with 1,2,3-trichlorobenzene was analysed
using the microtiter plate resting cell activity assay. Resting cell suspensions of microcosms enriched
over four transfers with 1,2,3-trichlorobenzene were tested for activity with brominated aromatic
electron acceptors. Activity was detected with all tested brominated compounds. Highest activity was
obtained with 2,4,6-tribromophenol and lowest activities with 3-bromo-2,4-dimethoxybenzoic acid
(Figure 3-10, left). To test whether the result of the microtiter plate activity assay can be transferred to
cultivation, twenty five microcosms from the coast off Chile previously cultivated with 1,2,3trichlorbenzene during four transfers were amended with an initial concentration of 5 µM 1,2,4tribromobenzene. 1,2,4-Tribromobenzene was selected despite higher activity detected with 2,4,6tribromophenols in activity assays, because 2,4,6-tribromophenol was observed to have negative
effects on cell growth in cultivation with strain CBDB1 (see also 3.1.5). Only one out of the 25
cultures showed activity with 1,2,4-tribromobenzene. 1,2,4-Tribromobenzene was transformed to 1,3-
57
Results
40
0.012
0.010
concentration (µM)
-1
activity (nkat ml )
30
0.008
0.006
0.004
20
10
0.002
0.000
0
NC TeCB -TBB MBA BPh BPh TBPh
4
-D -D 3,4 1,2, -3,5-D 2,4 2,6 2,4,6
,
2
1,
B
4-
0
electron acceptor
20
40
60
80
100
time (days)
Figure 3-10: Activity of resting cells from marine sediment microcosms with halogenated aromatic compounds.
Left: Microcosms enriched with 1,2,3-trichlorobenzene in the microtiter plate activity assay with brominated
aromatic electron acceptors, using 1,2,3,4-tetrachlorobenzene as positive control and acetone as negative control.
Cells were used directly from culture without concentration procedure and methyl viologen oxidation in the
negative control was subtracted from all samples. Right: Cultivation of a marine microcosm with 1,2,4tribromobenzene ( ) and production of 1,3- and/or 1,4-dibromobenzene ( ) and monobromobenzene ( ).
Arrows indicate when additional doses of 1,2,4-tribromobenzene were added to the microcosm. Abbreviations:
NC – negative control, TeCB – tetrachlorobenzene, TBB – tribromobenzene, 4-B-3,5-DMBA – 4-bromo-3,5dimethoxybenzoic acid, DBPh – dibromophenol, TBPh – tribromophenol.
and/or 1,4-dibromobenzene. The active microcosms was amended with additional doses of 20 µM
1,2,4-tribromobenzene after 41 days and 35 µM after 71 days. A small amount of monobromobenzene
was detected after 84 days of incubation (Figure 3-10, right). The detected loss of brominated
benzenes during the cultivation period of 120 days was possibly due to a frequently pierced rubber
septa from sample withdrawal during cultivation with 1,2,4-tribromobenzene and during previous
cultivation with 1,2,3-trichlorobenzene. Additionally, the headspace was frequently diluted by addition
of hydrogen and nitrogen to maintain a pressure of 1.4 bar within the serum bottles after samples
withdrawal.
3.4.2
Characterization of dehalogenation activity in marine sediments
To characterize the dehalogenation activity observed in the marine microcosms further,
experiments were carried out including oxygen and temperature sensitivity studies and specific
inhibition of cobalamin-dependent enzymes using propyl iodide. The tests for characterization of
dehalogenation activity were carried out in HPLC reaction vials. This allowed for the application of a
larger volume of resting cell suspension and reaction mix and thereby the detection of very small
changes of activity rates.
58
Results
Dehalogenation activity of resting cells from marine sediments exposed to oxygen was not
reduced compared to resting cells incubated in the anoxic glove box. To tests this, cells and medium
from actively dehalogenating microcosms were removed from the serum bottles and incubated either
in the anoxic glove box or outside the glove box under oxic conditions for up to four hours. At time
zero and after every hour, triplicates of samples incubated under anoxic conditions and triplicates of
samples incubated under oxic conditions were amended with activity assay reaction mix containing
approximately 350 µM 1,2,3,4-tetrachlorobenzenes and were incubated for 20 hours at 30°C under
anoxic conditions. The chlorinated benzenes were extracted with hexane from the activity assay
reaction mix and analysed by GC-FID. 1,2,3,4-Tetrachlorobenzene was transformed to 1,2,4trichlorobenzene by the resting cell suspensions. Dehalogenation activity decreased with increasing
incubation time after extraction from the microcosm serum bottles. Dehalogenation activity decreased
at similar rates in samples incubated under oxic or under anoxic conditions (Figure 3-11, left).
Dehalogenation activity in the marine microcosms at different temperatures was investigated.
Temperatures of 2°C have been reported for marine sediments (40). For the marine microcosms
highest activity of resting cells was observed at 30°C but low dehalogenation activity was also
detected at 4°C and at 60°C (Figure 3-11, centre). This was shown by incubating resting cells of the
marine microcosms with activity assay reaction mix and 1,2,3,4-tetrachlorobenzene at 4°C, 30°C and
60°C for 2 or 5 hours and analysis of chlorobenzenes by GC-FID.
100
50
80
40
1,2,4-TCB concentration (µM)
1,2,4-TCB production (µM)
Dehalogenating activity most likely originated from cobalamin-dependent enzymes such as reductive
60
40
20
0
20
15
30
10
20
5
10
0
0
0
1
2
time (h)
3
4
4
30
60
temperature (°C)
control
PI
PI + light
experimental set-up
Figure 3-11: Characterization of dehalogenating activity in marine sediments microcosms of the coast off Chile.
Left: Effect of oxygen exposure onto dehalogenation activity. 1,2,4-TCB was produced from 1,2,3,4-TeCB by
resting cell suspensions incubated for up to five hours under anoxic (dark grey bars) or oxic (light grey bars)
conditions. Centre: Temperature dependence of dehalogenation activity. 1,2,4-TCB production from 1,2,3,4TeCB by resting cell suspensions during incubation for 2 h (light grey bars) or 5 h (dark grey bars) at 4°C, 30°C
or 60°C.Right: Specific inhibition of cobalamin-dependent enzymes. 1,2,4-TCB produced from 1,2,3,4-TeCB in
the control compared to 1,2,4-TCB production by cells exposed to propyl iodide or propyl iodide and light.
Abbreviation: PI – propyl iodide. Shown values are means of triplicates +/- SD.
59
Results
dehalogenases (93, 94, 105, 109, 110). This was shown by activity assays with resting cells incubated
with propyl iodide (see also section 2.5.5), specifically and reversibly inhibiting cobalamin-dependent
enzymes. The resting cell suspensions containing 1.5 x 107 cells ml-1 were incubated with 10 µM
propyl iodide dissolved in acetone. Control set-ups contained resting cells and acetone. Another
control with resting cells was exposed to a light source after incubation with propyl iodide. Resting
cell suspensions were subsequently amended with 800 µl activity assay reaction solution, containing
100 µM 1,2,3,4-tetrachlorobenzene and incubated for 20 h at 30°C in HPLC-vials. The
1,2,4-trichlorobenzene production from 1,2,3,4-tetrachlorobenzene of resting cells incubated with
propyl iodide decreased compared to controls with resting cells previously incubated with acetone.
The exposure of resting cells incubated with 10 µM propyl iodide to light partially restored activity
(Figure 3-11, right).
3.4.3
Dehalogenation activity in Chloroflexi-containing sediments
Dehalogenation activity in marine sediments was investigated using in vitro activity assays and
sediments and crude extracts of marine sediments of the Baffin Bay core 371 and the Bay of Aarhus
and tidal flat sediments of the Wadden Sea (see also Table 2-3). Parallel studies in the Microflex
research project investigated occurrence and abundance of Dehalococcoidia by quantitative PCR.
Sediments of the Aarhus Bay and the Baffin Bay core 371 were shown to range from 1 x 105 to 1 x 107
Dehalococcoidia cells g-1 sediment through the depth of the 3 or 4 m core, respectively (47). Tidal flat
sediments were shown to contain 1 x 104 to 1 x 105 Dehalococcoidia cells g-1 sediment in the surface
sediments of 1-8 cm (47) (see also Table 2-3). Experiments conducted in the current study showed that
a minimum of ~3 x 105 cells ml-1 of Dehalococcoidia strain CBDB1 were required to detect
dehalogenation activity in the in vitro assays. To obtain a cell number of at least 3 x105
Dehalococcoidia cells ml-1 from sediments, cells of up to 30 g sediments were extracted by Nycodenz
and density centrifugation (see also 2.4.4). Obtained cells were subsequently dissolved in 500 µl
cultivation medium or physiological NaCl solution and were subsequently added as crude cell
suspensions to the activity assay in the microtiter plate or in HPLC-vials. Additionally, sediment
slurries of 1 to 10 g sediments from various depths were added directly to the activity assay solutions.
Dehalogenation activity was tested with 1,2,3-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, 1,2,4tribromobenzene or 2,4,6-tribromophenol. Activity assays conducted in HPLC-vials sediments or cell
suspension were incubated for up to 72 hours at 30°C and the halogenated compounds were then
extracted with hexane for analysis by GC-FID. Dehalogenation activity was neither detected by
photometer-based nor by GC-FID-based methods.
60
Results
3.5
Chemical properties of halogenated aromatic compounds influence reductive
dehalogenation patterns
Experiments conducted with strain CBDB1 in the current study and in previous studies
demonstrated a wide range of halogenated molecules are transformed by D. mccartyi strain CBDB1
(7, 119, 121, 124, 193, 194). The multitude of halogenated compound types and congeners
transformed by strain CBDB1 exceeds the number of reductive dehalogenases reported in the genome
of strain CBDB1 (26), providing evidence that a single reductive dehalogenase is able to transform
more than one single congener. This was supported by in vitro activity assays and reductive
dehalogenase expression studies in the current study. However, the reasons for this electron acceptor
diversity are poorly understood. For the different tested compound classes specific dehalogenation
patterns were observed in the current and in previous studies (7, 121) and chemical properties of the
halogenated compound seem to have an important influence onto dehalogenation pathways. For
instance, isolated bromine substituents were dehalogenated in activity assays by strain CBDB1 cells
previously grown with 1,2,3-trichlorobenzene, while isolated chlorine substituents of 1,3dichlorobenzene are not dehalogenated. In addition, the same was observed for dehalogenating
microorganisms from marine sediments which were able to dehalogenate isolated bromine substituents
but not chlorine substituents suggesting that the chemical properties of a halogenated electron acceptor
are as important as the enzymatic properties of the dehalogenating strain. Therefore, chemical
properties of 58 halogenated molecules were evaluated in detail (Table 7-1 to Table 7-16) for their
ability to predict experimentally observed dehalogenation pathways of strain CBDB. This was done to
gain a deeper understanding of the mechanism of reductive dehalogenation which allows reductive
dehalogenases to transform a wide spectrum of halogenated electron acceptors and to better
understand which parameters explain the different degree of dehalogenation observed for molecules
with different functional groups and halogen types. For all analysed molecules experimental data with
strain CBDB1 was available. The findings possibly will allow also for an extrapolation onto properties
generally required for halogenated compounds to serve for organohalide-respiring Dehalococcoidia as
electron acceptor and even for dehalogenation microorganisms from different bacterial classes but will
need to be tested and also correlated to enzymatic properties in future studies.
Several chemical descriptors were chosen, which were described previously to depict chemical
properties of a molecule such as reactivity or electron density distribution (195). The theoretical
descriptors analysed in the current study included HOMO/LUMO, electrophilicity, local
electrophilicity, partial charges for the neutral molecule and partial charges of the anionic form of a
molecule. The selected chemical descriptors were modelled by Dominik Wondrousch within a
cooperation project with the Ecological Chemistry Department of the UFZ. The modelled data were
evaluated in the current study for their ability to predict the regioselective removal of halogens during
organohalide respiration of strain CBDB1 and to allow for an analysis of the influence of functional
61
Results
groups onto halogen substituents. In the current study only the chemical descriptors which revealed to
successfully predict dehalogenation patterns of strain CBDB1 are shown and discussed.
3.5.1
The most positive halogen atomic net charge predicts regioselective dehalogenation
by strain CBDB1
Three different partial charge models, the Mulliken (125, 187), the Hirshfeld (188) and the NBO
model (196, 197) were analysed for their ability to predict dehalogenation pathways of halogenated
compounds catalysed by strain CBDB1 (Table 7-1 to Table 7-12). For the dehalogenation pathway
prediction only electron acceptors with identified reaction pathways were included. Electron acceptors
were not included into the analysis, when all halogen substituents are equally likely to be
dehalogenated. Examples for such molecules are hexachlorobenzene and dibromobenzenes. Also
pentachlorophenol was omitted from the prediction, because the reaction pathways of
pentachlorophenol catalysed by strain CBDB1 could not be resolved experimentally (119).
For the prediction of dehalogenation pathways catalysed by strain CBDB1, the data were
evaluated according to the following hypotheses: a) the halogenated carbon with the most positive
partial charge predicts regioselective dehalogenation, b) the halogenated carbon with the most
negative partial charge predicts regioselective dehalogenation, c) the halogen with the most positive
partial charge predicts regioselective dehalogenation and d) the halogen with the most negative partial
charge predicts regioselective dehalogenation.
Halogenated phenols were evaluated as non-dissociated neutral molecules and also as dissociated
phenolate molecules. It is not clear whether D. mccartyi strain CBDB1 uses phenols, phenolates, or
both as electron acceptor. At 25°C in water, phenol and halogenated phenols are weak acids (examples
of pKa values: phenol 9.99, 4-chlorophenol 9.41, 2-chlorophenol 8.56, 2,3-dichlorophenol 7.44, 2bromphenol 8.25, 4-bromphenol 9.37), and are therefore ionized only to a small fraction. For the
evaluation of partial charges, phenolates have the advantage of symmetry in geometry optimization in
contrast to phenols, which contain the non-dissociated hydroxy group. Quantum mechanical methods
Figure 3-12: Example for the effect of the hydroxy group on the partial charge modelled for halogens for 2,4,6dibromophenol and 2,4,6-dibromophenolate, shown for NBO halogen atomic net charges. A) 2,4,6tribromophenol, halogen substituents in ortho-position with the modelled partial charge B) 2,4,6tribromophenol, halogen substituents in ortho-position with averaged partial charge C) 2,4,6-tribromophenolate,
halogen substituents with the modelled partial charge.
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Results
distinguish between conformers that under natural conditions represent the same molecule (for
instance 2-bromophenol and “6-bromophenol”) depending on the modelled orientation of the hydroxy
group and the intramolecular interactions of the hydroxy hydrogen with neighbouring halogens (198).
The asymmetry of the modelled hydroxy group influences therefore the modelled partial charges of
flanking halogens (Figure 3-12) and thereby the correct prediction of dehalogenation pathways. This
effect could be counteracted by averaging the partial charge value of hydroxy group flanking
halogens; however this approach can be applied only to symmetrically substituted molecules such as
2,4,6-tribromophenol but not to asymmetric substituted molecules such as 2,3,6-tribromophenol.
Therefore, for each model the partial charge data were evaluated taking into account once the
dissociated phenolates and once the non-dissociated phenols.
All analysed models gave best prediction for dehalogenation pathways using the most positive
halogen atomic net charge as indicator (Table 3-5 A and B). Second best prediction rates were obtained using the most negative atomic net charge of halogen-substituted carbons (Table 3-5 A and B).
Figure 3-13: Calculations of partial to partial charges to predict the dehalogenation pathway of 2,4,6tribromophenol by D. mccartyi CBDB1 A) Mulliken model, B) Hirshfeld model C) NBO model.
63
Results
When using the most positive partial halogen charge, the Mulliken partial charge system revealed
good prediction rates of dehalogenation pathways catalysed by strain CBDB1 for most molecules
except for phenols and 3-bromo-2-chloropyridine (Supplementary Table 7-1 to Table 7-4).
A correct prediction of the dehalogenation pathway of phenols using the most positive halogen
partial charge was hampered by the influence of the asymmetric hydroxy group onto the
ortho-substituted halogens (Table 3-5 A, see also Figure 3-12). Therefore, improved prediction rates
were obtained when phenolates instead of phenols were included into analyses (Table 3-5 B,
Supplementary Table 7-1 and Table 7-4).
For Hirshfeld partial charges, the prediction was more successful when including phenols instead
of phenolates into analyses (Table 3-5 A and B, and Supplementary Table 7-5 to Table 7-8). In
addition, Hirshfeld charges did not predict correctly the dehalogenation of 3-bromo-2-chloropyridine
when using the halogen with the most positive atomic net charge.
Table 3-5: Rates of successful prediction of dehalogenation products by D. mccartyi strain CBDB1 using partial
charges. For the evaluation of the prediction rate 27 molecules were used. Phenols were analysed in the nondissociated (A) and in the dissociated form (B).
A
Indicator
Most positive C
Most negative C
Most negative X
Most positive X
Delta X/C
Mulliken model
Det. TP
Pref. TP
28%
68%
68%
20%
88%
80%
84%
Hirshfeld model
Det. TP
Pref. TP
24%
80%
76%
8%
96%
92%
4%
NBO model
Det. TP
Pref. TP
16%
76%
72%
24%
88%
84%
88%
B
Indicator
Most positive C
Most negative C
Most negative X
Most positive X
Delta X/C
Mulliken model
Det. TP
Pref. TP
28%
88%
88%
8%
96%
92%
80%
Hirshfeld model
Det. TP
Pref. TP
20%
64%
60%
20%
80%
76%
36%
NBO model
Det. TP
Pref. TP
20%
92%
84%
8%
96%
92%
84%
The carbon charge refers only to carbon atoms substituted by a halogen. X = halogen atomic net charge, C =
carbon atomic net charge. Delta X/C = Prediction rates were calculated using the most pronounced difference
between the halogen partial charge and the carbon partial charge. Det. TP = Detected transformation product.
Prediction rates were calculated from the number of correct prediction of the exact transformation product
detected in cultivation experiments with strain CBDB1; Pref. TP = Preferential transformation product.
Prediction rates were calculated from the number of correct predictions of the most abundant transformation
product of strain CBDB1 compared to other possible transformation products. Therefore the Pref. TP rate can be
the same or smaller than the Det. TP.
For NBO atomic net charges, improved prediction rates were obtained when including phenolates
instead of phenols into analyses (Table 3-5 A and B, and Supplementary Table 7-9 to Table 7-12).
Improved prediction results were obtained for symmetrically substituted phenols when the net atomic
halogen charge was averaged (Figure 3-13). The NBO partial charge model allowed for a correct
prediction of the dehalogenation pathway of 3-bromo-2-chloropyridine, as a molecule substituted with
bromine and chlorine. Strain CBDB1 debrominated 3-bromo-2-chloropyridine, but it was not able to
dechlorinate 3-bromo-2-chloropyridine. The NBO model assigned a more positive partial charge to
64
Results
bromine than to chlorine, corresponding to a higher electronegativity of chlorine compared to
bromine.
3.5.2
Sigma NBO halogen partial charges predict regioselective dehalogenation
The NBO partial charge model allowed for the modelling of the separate sigma and pi
contributions to the partial charge of an atom. The NBO sigma and pi partial charges were modelled
by Dominik Wondrousch (Department Ecological Chemistry, UFZ Leipzig) (Supplementary Table
7-13 to Table 7-16). Improved prediction rates when using sigma partial charges compared to pi
partial charges indicated an increased importance of sigma electron withdrawal from the halogen by
negative inductive effects of neighbouring substituents for reductive dehalogenation. This conclusion
was drawn from analyses of the halogen sigma and pi partial charges for halogenated heteroaromatic
and homoaromatic compounds tested in the current study. For this, sigma-binding electron
contributions and pi-binding electron contributions of the halogen with the most positive partial charge
were analysed for their ability to reflect the dehalogenation pathways catalysed by strain CBDB1. The
halogen with the most positive partial charge was used for this evaluation because it was identified in
the current study as the most successful indicator for the dehalogenation pathway prediction of strain
CBDB1. The analysis of sigma and pi partial charges revealed different prediction rates for
regioselective dehalogenation by strain CBDB1. When using the most positive halogen sigma partial
charge 96% and 92% of the experimentally observed dehalogenation pathways were predicted
correctly, when including phenolates or phenols into analyses, respectively. The most positive halogen
pi partial charge predicted 76% or 60% of regioselective removal of halogens by strain CBDB1, when
including phenols or phenolates into analysis, respectively.
3.5.3
Analysis of the influence of different secondary substituents onto the halogen atomic
net charges
By evaluating atomic net charges of different atoms within a molecule as an indicator for
regioselective removal of halogens during organohalide respiration, the halogen with the most positive
atomic net charge was identified as the indicator with highest successful prediction rate. Moreover,
improved dehalogenation pathway predictions using sigma partial charges compared to pi partial
charges indicated withdrawal of sigma-binding electrons from the halogen as important parameter for
dehalogenation. Considering this, the partial charges of halogens flanking non-halogen substituents
and the halogen-substituted carbon net charges in ortho-position to the non-halogen substituent were
compared. Because NBO partial charges allowed for the comparison of sigma and pi contributions to
the partial charge, NBO charges were used for this analysis. The non-halogen substituents of 2,6dichloroanilin, 2,6-dichlorophenol, 2,6-dichlorophenolate and 2,6-dichlorobenzonitrile induced
increasingly positive partial charges in the flanking halogen substituents in the order of -O- < -NH2 < OH < -CN (Figure 3-14, A). This corresponds to the different degree of reductive dehalogenation of
65
Results
2,6-dichloroaniline, 2,6-dichlorophenol and 2,6-dichlorobenzonitrile observed experimentally. 2,6dichloroaniline is not dehalogenated by strain CBDB1 (see also 3.1.1), 2,6-dichlorophenol is
dehalogenated only partially and incomplete (119) and 2,6-dichlorobenzonitrile is dehalogenated to a
non-halogenated end product (199). An experimental distinction of dehalogenation of phenolates
compared to phenols by strain CBDB1 is so far not available. When including 1,2,3-trichlorobenzene
into analysis, the halogen atomic net charge places the influence of a halogen substituent in orthoposition with an additional halogen substituent in meta- and para- position in the following
order: -O < -NH2 < -OH < -Cl < -CN.
sigma
pi
total
0.4
A)
0.0
0.0
-0.1
-0.2
-0.2
-0.4
2/6 l 2/6
2/5 l 2/5
C
C
C
h
y
h
C
C P - D C T D CT
D
D
2,6
2,5
2,6
2,5
2/6 l 2/6 l 2/6 l 2/6
Cl
C
C
C
e
t
A
N
Ph
a
P h ,6-DC -DC -D CB
C
6
,
2
2
-D
2,6
2,6
Py
C
atom
atom
0.6
sigma
pi
total
0.2
C)
atomic net charge
0.4
atomic net charge
B)
sigma
pi
total
0.2
0.1
atomic net charge
atomic net charge
0.2
0.2
0.0
-0.2
D)
sigma
pi
total
0.1
0.0
-0.1
-0.4
-0.6
-0.2
6
2/6 r 2/6
2/6
2/
C
Cl
C
B
h
h
h
P
P
P
Ph
B
C
C
-D 2,6-D ,6-D B
-D
6
6
,
,
2
2
2
2
2
6
6
F
C
Cl
N
N
N
BN
B
B
F
B
F
F
F
-6
-6- C-6-62-C
2-C
2-C
2C
atom
atom
Figure 3-14: Halogen (QX) and halogen-substituted carbon (QC) atomic net charges and their sigma and pi
binding electron contributions of selected molecules. A) QX of 2,6-chlorophenolate, -aniline, phenol, and
benzonitriles. B) QX and QC of 2,6-dichloropyridine and 2,5-dichlorothiophene; C) QX and QC of 2,6dichlorobenzonitrile and 2-chloro-6-fluorobenzonitrile. D) QX and QC of 2,6-dichlorophenol and 2,6dibromophenol.
66
Results
3.5.4
The influence of chemical properties of heterocycles onto reductive dehalogenation
Halogen and carbon atomic net charges of electron-rich heteroaromatics, such as the fivemembered thiophene, were compared with halogen and carbon atomic net charges of the electron-poor
pyridine. 2,3- and 2,6-dichloropyridine were not dehalogenated while 2,3- and 2,5-dichlorothiophenes
were dehalogenated (see also section 3.1.4 and 3.3.1). When comparing chlorine atoms flanking the
heteroatoms of 2,6-dichloropyridine or 2,5-dichlorothiophene a more positive partial charge for
halogens of 2,5-dichlorothiophene than for halogens of 2,6-dichloropyridine was observed. Moreover,
atomic net charges of the halogen-substituted carbons of pyridine had more positive atomic net
charges than halogen-substituted carbons in 2,5-dichlorothiophene (Figure 3-14, B). Furoic acids were
not compared with thiophenes and pyridines because all brominated aromatic congeners were
debrominated by strain CBDB1 regardless of the molecular structure tested.
3.5.5
The role of the halogen type in reductive dehalogenation
When comparing atomic net charges of fluorine and chlorine in 2-chloro-6-flurobenzonitrile all
models assigned fluorine a more negative atomic net charge than chlorine (Figure 3-14, C). The
atomic net charge of the fluorine-substituted carbon was more positive than the atomic net charge of
the chlorine-substituted carbon. Although for the fluorine-substituted carbon very positive atomic net
charges were calculated, no dehalogenation of fluorine was observed underscoring the importance of
the halogen charge for reductive dehalogenation compared to the carbon charge. For the halogen
partial charge, the sigma partial charge contribution differed more between chlorine and fluorine than
the pi partial charge contribution. When comparing chlorine and bromine in 2,6-dibromophenol
and2,6-dichlorophenol, more positive partial charges for bromine than for chlorine were modelled.
The sigma-binding electrons contributed thereby more to the differences of the total halogen net
charge than the pi-binding electrons (Figure 3-14, D). This underscores the importance of withdrawal
of sigma binding electrons from the halogen for reductive dehalogenation, i.e. by secondary
substituents with negative inductive effects to counteract the negative inductive effect exhibited by the
halogen itself.
3.5.6
Correlation of the most positive halogen partial charge with resting cell activity
Partial charge of the most positive halogen substituent were analysed for a correlation with
specific activities measured in resting cell activity assays. No correlation was observed when
comparing all experimentally tested molecules with each other. However, when only one compounds
group with the same halogen type was compared, more positive halogen partial charge values were
paralleled with higher activity in resting cell activity assays (Figure 3-15), however no linear
correlation was observed.
67
Results
100
Chlorothiophenes
Bromobenzenes
Chlorophenolates
Bromophenolates
activity (nkat mg-1)
80
60
40
20
0
-0.15 -0.10 -0.05
0.00
0.05
0.10
0.15
partial charge
Figure 3-15: Correlation of specific activities of strain CBDB1 with halogen atomic net charges. Shown are the
means of triplicate measurements from three different microtiter plate activity assays ± SD. Within each
compound class higher activity was observed the more positive atomic net charges were on the halogen atom.
3.6
Non-halogenated compounds as potential respiratory electron acceptors for
Dehalococcoidia
In this chapter, the investigation of non-halogenated electron acceptors of Dehalococcoidia by
molecular methods is described. Analysis of the genomes of a single Dehalococcoidia cell
denominated “DEH-J10” isolated from marine sediments of the Bay of Aarhus (46) and from two
single cells “Dsc1” and “Dsc2” from the Peruvian margin (48) revealed the potential of
Dehalococcoidia subgroups to grow by an organohalide respiration-independent mode of living. In a
further genome of a Dehalococcoidia single amplified genome denominated “SAG-C11”, a gene
coding for an RdhA subunit was identified. However, a TAT leader sequence and a gene coding for
the putative membrane anchor protein RdhB were missing (personal communication, Dr. Kenneth
Wasmund, UFZ, Leipzig) suggesting the RdhA is not a functional, membrane-bound respiratory
reductive dehalogenase. Beside the gene coding for RdhA, several genes potentially involved in
respiratory processes were identified in the genome, including an operon coding for proteins involved
in dissimilatory sulphite reduction (personal communication, Dr. Kenneth Wasmund, UFZ, Leipzig).
The region contained dsrA, dsrB, dsrD, dsrN, dsrC, dsrM and dsrK genes. Downstream of the dsrregion, genes were detected which showed highest similarity to genes described in Dehalogenimonas
lykanthroporepellens involved in siroheme synthesis. Upstream of the dsr-region a gene coding for a
two-component transcriptional regulator luxR was identified. This gene has its most similar homologs
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Results
in the Chloroflexi species Dehalogenimonas lykanthroporepellens BL-DC-9 and Ktedonobacter
racemifer (Accession number EFH84403) (Figure 3-16).
Dissimilatory sulphite reductase genes were described to occur in a restricted number of bacterial
phyla (150) but have never been described to occur in the phylum Chloroflexi. In addition, many
dissimilatory sulphite reductase sequences of so far uncultured bacterial species remain with unknown
phylogeny. Therefore, the diversity and occurrence of dissimilatory sulphite reductase genes linked to
the phylum Chloroflexi were investigated in the current study. This may contribute to the
understanding of phylogenetic diversity of dissimilatory sulphite reductase genes in public databases
and the metabolic potential harboured by the class Dehalococcoidia. The distribution and diversity of
dsr genes linked to the class Dehalococcoidia was investigated using molecular methods, because
previous attempts in the current study to approach this research question by cultivation remained
unsuccessful (data not shown).
DNA fragments coding for dsr genes and other genes that could provide a phylogenetic link for
the determination of the DNA source organism were amplified to link dissimilatory sulphite
reductases from marine sediments to Dehalococcoidia. The 16S rRNA gene sequence was not
encoded in the close genetic environment of dsr related genes in SAG-C11. Therefore, a coamplification of the dsr genes and the 16S rRNA gene sequence was not possible. Therefore, genes
detected in DEH-SAG-C11 with highest similarity to genes of Dehalogenimonas lykanthroporepellens
encoded adjacent to dsr genes were used as such markers. Another approach was to amplify longrange PCR fragments coding for several dsr related genes. This allowed for phylogenetic analysis of
several new dsr genes and a comparison of their sequence order with the sequence order of the dsr
coding region of DEH-SAG-C11. For this purpose primers were designed to amplify dsr genes with
similarity of the dsr locus in single cell SAG-C11 in a long-range PCR (Figure 3-16), sediments for
DNA extraction were sampled or selected and methods for DNA extraction suitable for long-range
PCR approaches were tested.
Figure 3-16: Dissimilatory sulphite reductase genes dsrABDNCMK (black arrows) and flanking genes with
highest similarity to genes occurring in Dehalogenimonas lykanthroporepellens BL-DC-9 (dark grey arrows)
associated with siroheme synthesis (cobA/hemD, hemC, hemA and cysG) or transcriptional regulation (luxR)
detected in Dehalococcoidia SAG-C11 and selected primer binding positions.
69
Results
3.6.1
Selection of sediment cores and cell extraction
Sediments were selected for extraction of cells and DNA to investigate the distribution of dsr
genes possibly linked to Dehalococcoidia. A 3 m sediment core was recovered from the same location
of the Aarhus Bay which allowed for the isolation of SAG-C11 to have large quantities of SAG-C11
phylotype-containing sediment for experimental work. At the top of the sediment core sulphate had a
concentration of 31.7 mM. The sulphate concentration decreased constantly to ~2 mM at 171
centimetres below sea floor (cmbsf), and remained at this concentration down to 304 cmbsf. The
concentration of 2 mM sulphate was attributed to sulphide reoxidation, which might have occurred
during sample handling and preparation for shipment of the sediment core. Previous geochemical
studies of the same sediment site suggest a sulphate concentration of 0.2 mM from 150 cmbsf
downwards (152). To obtain DNA in sufficient quality for long-range PCR applications two
approaches were used including a cell extraction by density centrifugation using Nycodenz or by
direct DNA extraction using the FastDNATM Spin Kit for Soil. Nycodenz had the advantage that cells
could be separated from soil to subsequently apply DNA isolation methods which have been reported
to reduce sheering of DNA (see also section 2.7.1). Additionally, higher amounts of sediments could
be extracted compared to the FastDNATM Spin Kit for Soil, which allows for the extraction of 0.5 g of
sediment per provided reaction container.
Cells were extracted from sediments of the Bay of Aarhus from 10–20, 30–36, 66–72, 102–108,
135–141, 168–174, 201–208, 235–341, 268–274, and 301–308 cmbsf using 80% w/v Nycodenz or the
FastDNATM Spin Kit for Soil. Cell extraction from 30 g of sediments of 10–20 cmbsf using Nycodenz
yielded 1.9 ± 0.3 to 2.5 ± 0.5 x 108 cells ml-1, after resuspending the cell pellet in 500 µl 0.9% NaCl
solution. Second extraction steps of the same sediment samples yielded up to 7.8 ± 2.6 x 106 cells ml-1.
Additionally, sediments from the Baffin Bay sampled by Camelia Algora during the Polarstern
cruise 2010 were included, as a sediment sample from deep-sea sediments (see also Table 2-4 for
more details of the sampling sites). Core 371 was selected, due to high copy numbers of dsrA genes
Figure 3-17: Epifluorescence microscopic pictures (185 µm x 148 µm) of cells extracted from 30 g marine
sediments with 80% Nycodenz solution resuspended in 500 µl 0.9% NaCl solution and stained with SYBR
Green. Left: Cells extracted from tidal flat sediments of 10–20 cmbsf of the Wadden Sea resulting in 1.7 x 108
cells ml-1. Middle: Cells extracted from Aarhus sediments of 0–20 cmbsf resulting in 2.5 x 108 cells ml-1. Right:
cells from Baffin Bay sediments of 280–300 cmbsf resulting in 5.2 x 107 cells ml-1.
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Results
determined previously (200), a similar distribution of Chloroflexi sub-groups in the sulphate zone of
Baffin Bay core 371 compared to the core sampled in the Bay of Aarhus and the presence of the
SAG-C11 Dehalococcoidia subgroup DSC-GIF3-B throughout the sediment core (47). Sediment
samples were not available for all depths of the core due to previous experiments in different research
projects. Therefore, cells and DNA were extracted only from sediments of 280 and 300 cmbsf. A first
extraction of cells from 30 g of sediments from 280 cmbsf using Nycodenz typically yielded 5.2 ± 0.6
x 107 cells ml-1 when the cell pellet was resuspended in 500 µl 0.9% NaCl solution. A second
extraction of cells from the same sediment sample yielded additional 6.1 ± 0.3 x 106 cells. The
extraction of cells from sediments of 300 cmbsf yielded around 9.5 ± 0.3 x 106 cells ml-1 and a second
extraction of cells from the same sediments yielded 1.3 ± 1.4 x 106 cells ml-1 (Figure 3-17).
Tidal flat sediments from the Wadden Sea, sampled by Anton Schulte-Fischedick, were included
to investigate the presence of dissimilatory sulphite reductase genes linked to Chloroflexi in another
marine habitat. Previous studies showed the occurrence of Dehalococcoidia in these sediments (47).
Tidal flat sediments from the Wadden Sea were available from upper layers of 1–20 cmbsf. Cell
extractions using Nycodenz yielded between 5.7 ± 0.8 x 107 to 1.7 ± 0.4 x 108 cells ml-1 when
resuspending the cell pellet in 500 µl 0.9% NaCl solution and second extractions of cells from the
same sediments yielded up to 3.7 ± 1.2 x 107 cells ml-1.
Sediment samples from the Peru Margin and the Black Sea were available to investigate
dissimilatory sulphite reductase genes linked to Chloroflexi (Table 2-3). However, sediments of the
Peru Margin and the Black Sea were available only in very low quantities and poor quality. DNA was
extracted using the FastDNATM Spin Kit for Soil.
3.6.2
Selection of fragments for analysis
At first, attempts were made to amplify a ~10 kb fragment coding for dsrABDNCMK and genes
phylogenetically related to genes from Dehalogenimonas lykanthroporepellens using specific and
degenerated primers targeting a highly conserved region of dsrA and SAG-C11 specific region of
hemA. The forward primer Dsr1F-DHC-2-pt targeting dsrA was based on the primer sequence used by
Wagner and colleagues (151) and was modified to match the sequence of SAG-C11. It was combined
with one of six different reverse primers with different binding positions in hemA, different degrees of
degeneracy or different 3-prime-end-modifications to avoid degradation of primers by exonuclease
activity of the polymerase during long range PCR amplifications (for primer targets and sequences see
Table 2-10). However, while it was possible to amplify a ~10 kb fragment using genomic DNA of
SAG-C11 as template for polymerase chain reaction, it was not possible to amplify the 10 kb fragment
from DNA isolated from marine sediments. Instead, many lower molecular weight fragments were
amplified, which were not further investigated. The same was observed during amplification of a
~8 kb fragment coding for dsrABDNCMK and hemD, which yielded many lower molecular weight
fragments but not the targeted 8 kb fragment. The primers C11_luxr1_out1 and Sag_D_gammaF_C
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Results
were used for the amplification of a 4.6 kb fragment coding for luxR and dsrABDN (Figure 3-16). A
faint band with a size of 4.6 kb was amplified from DNA isolated from sediments of Aarhus, however,
purification and cloning remained unsuccessful. Attempts to improve the amplification of the 4.6 kb
fragment by touch-down PCR, additional purification steps of the template DNA used for
amplification or addition of different concentrations of DMSO and MgCl2 to the amplification
reactions did not enhance the amplification of the band.
Purification and cloning of fragments above 4 kb coding for dsr genes and a phylogenetic marker
gene remained unsuccessful, therefore, attempts to amplify a smaller 2.5 kb fragment were carried out
using primers LuxR_out1 and DsrR4_C11 without any degeneracy (Table 2-10). These primers target
dsrB and the transcriptional regulator sequence, which in SAG-C11 has highest similarity with luxR of
Dehalogenimonas lykanthroporepellens strain BL-DC-9. Amplification and cloning of a 2.5 kb
fragment amplified from shallow sediments of 10–20 cmbsf yielded twenty-two clones harbouring a
2.5 kb insert coding for dsrB and a putative transcriptional regulator of the arsR family (luxR) (Table
7-17). Most sequences were highly similar to dsrB and luxR of SAG-C11, however, several sequences
differed from drsAB and luxR genes of SAG-C11 (see for further discussion section 3.6.3). The
amplification of the 2.5 kb fragment coding for dsrAB and luxR using degenerated primers (Table
2-10) or the amplification of the 2.5 kb fragment from deeper sedimentary levels with specific primers
remained unsuccessful. Generally, the amplification of fragments of above 1 kb coding for dsr genes
and marker genes with highest similarities to Dehalogenimonas lykanthroporepellens strain BL-DC-9
from DNA isolated from marine sediments was difficult. Primers with low degeneracy amplified
sequences with very high sequence similarity to dsrAB genes of SAG-C11, and thus did not fulfil their
designated purpose for the analysis of sequence diversity of functional genes in the class
Dehalococcoidia. Primers with higher degeneracy amplified besides DNA fragments with the targeted
size a broad range of smaller fragments. Even after purification and cloning of fragments with the
targeted size, sequencing revealed a variety of different functional genes that were not targeted (data
no shown).
Different primer binding positions and primers with different degree of degeneracy were tested to
amplify dsr genes with higher diversity. The primer combinations Dsr1F-DHC-2-pt1 and
SAG_D_gammaF_A, Dsr1F-DHC-2-pt2 and SAG_D_gammaF_B and Dsr1F-DHC-2-pt3 and
SAG_D_gammaF_C or Dsr1F-DHC-2-pt2 and SAG_D_gammaF_C all based upon dsr sequences
identified in SAG-C11, target a fragment of ~3.9 kb (see also Table 2-10 and Figure 3-16). These
primers used in different combinations amplified many unspecific fragments of ~0.5 to ~6 kb and a
fragment of the targeted size of 3.9 kb, when using DNA isolated from sediment samples of Aarhus,
the Baffin Bay or the Wadden Sea. Purification and cloning of the 3.9 kb fragment amplified from
sediments of Aarhus from 10-20, 135-141, 168-174 and 268-274 cmbsf, from the Baffin Bay from
280–300 cmbsf and from tidal flat sediments from the Wadden Sea of 10–20 cmbsf, allowed for the
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Results
identification of twenty-six clones coding for dsrA and dsrN (Supplementary Table 7-18). No
fragments corresponding to a size of 3.9 kb were amplified from sediments of Peru and the Black Sea.
Obtained sequences will be submitted to NCBI sequence read archive (for SRA number see
Supplement).
3.6.3
Linking dissimilatory sulphite reductase genes with Dehalococcoidia
Dissimilatory sulphite reductase genes amplified from marine sediments in the current study were
investigated for their phylogenetic origin and potential relatedness to Dehalococcoidia. BlastX
searches with luxR and dsr sequences derived from marine sediments were carried out using the NCBI
nr database supplemented with the amino acid sequences of SAG-C11. Phylogenetic trees were
constructed with amino acid sequences of predicted open reading frames of dsrA, dsrB and dsrN
amplified in the current study and with amino acid sequences retrieved from the NCBI nr database.
Additionally, nucleotide and amino acid sequences were compared in global alignments for their
similarity to sequences detected in SAG-C11 (see also 2.8).
In the previous chapter the identification of twenty-two clones harbouring a 2.5 kb insert coding
for LuxR and DsrAB was described (Supplementary Table 7-17). BlastX searches of the LuxR
sequences derived from marine sediments revealed highest scores (and lowest e-values) for the LuxR
transcriptional regulator of Dehalococcoidia SAG-C11 confirming the specificity of the used primers
for Dehalococcoidia-related luxR genes. The next best hits belonged to the Chloroflexi strains
Ktedonobacter racemifer (DSM 44963) and Dehalogenimonas lykanthroporepellens BL-DC-9 or to
the Firmicutes strain Dehalobacter DCA (Supplementary Table 7-17). The high sequence diversity of
luxR transcriptional regulators in different bacterial groups did not allow for the construction of
reliable phylogenetic trees. Global alignments of the approximately 112 amino acid counting LuxR
sequences derived from sediments of the Aarhus Bay gave 93% and 100% sequence similarity to
LuxR of SAG-C11 (Supplementary Table 7-17). Sequence similarities of the global alignments of
luxR nucleotide sequences were between 96% and 100%. Amino acid sequence similarities to LuxR of
Ktedonobacter racemifer (DSM 44963) and Dehalogenimonas lykanthroporepellens BL-DC-9 or to
the Firmicutes strain Dehalobacter DCA were between 47% and 55% in local alignments
(Supplementary Table 7-17).
DsrB amino acid sequences amplified with the same fragment coding for LuxR derived from
sediments of the Aarhus Bay had highest BlastX scores and lowest e-values for DsrB of Chloroflexi
SAG-C11. All analysed DsrB sequences amplified on one fragment with luxR clustered with DsrB
sequence of SAG-C11 in phylogenetic analysis using the Neighbor Joining algorithm (Figure 3-18).
The DsrB sequence of clone AA169 was not included into phylogenetic analysis because the obtained
sequence was very short and the luxR sequences could not be analysed for this clone (see also Table
7-17).
73
Results
Branch colours Figure 3-18: Phylogenetic tree based on a global alignment of ~295 DsrB amino acid sequences using the
Neighbor Joining method implemented in Mega 6.0 with 1000 bootstrap tests and pairwise deletion,
evolutionary distance were computed with the p-distance method. Sequences amplified in the current study are
marked with a green caption. DsrB of SAG-C11 is highlighted with a green background color. Numbers show
bootstrap values above 0.5.
74
Results
Phylogenetic analysis by the Minimum Evolution, or the Maximum Likelihood algorithm showed
likewise to the Neighbor Joining algorithm, clustering of the DsrB sequences with DsrB sequences of
SAG-C11. When comparing sediment derived DsrB sequences of 70 to 238 amino acids with DsrB of
SAG-C11 in global alignments, sequence similarities of 97% to 100% were obtained (Supplementary
Table 7-17). Nucleotide sequences similarities were between 95% and 100% when compared to dsrB
of SAG-C11 (Supplementary Table 7-17).
Additionally, nineteen clones harbouring a 3.9 kb insert derived from sediments of the Bay of Aarhus
and coding for dsrA and dsrN were analysed (Supplementary Table 7-18). BlastX analysis of the
obtained dsrA sequences revealed highest scores and lowest e-values for DsrA of Dehalococcoidia
SAG-C11 or uncultured sulphate reducers (Supplementary Table 7-18). Phylogenetic analysis of the
DsrA sequences from Aarhus sediments showed ten DsrA sequences encoded on the 3.9 kb fragment,
clustered with DsrA of SAG-C11 (Figure 3-19). Short DsrA sequences of clones AA238 and AA139
were not included into phylogenetic analysis. DsrA sequences of 87 to 386 amino acids revealed 83%
and 99% sequence similarities when compared to DsrA of SAG-C11 in global alignments. The
respective nucleotide sequence similarities ranged from 76% and 98% (Supplementary Table 7-18).
All sequences amplified from sediment layer below 10–25 cm and two DsrA sequences from shallow
sediments of Aarhus (clones Aarhus clone 271 and Aarhus clone 252), affiliated with sequences
previously termed ‘uncultured DsrAB lineage 3’ (201) (Figure 3-19) shown to become more prevalent
with increasing depth and lower sulphate concentrations in sediments (152). DsrA sequences
amplified from deeper sedimentary levels had 75% to 81% amino acid sequence similarity in global
alignments with DsrA of C11 (87 to 386 amino acids), and nucleotide similarity was between 72% and
75%.
Although several DsrA amino acid sequences did not cluster with DsrA of SAG-C11, their DsrN
sequences amplified within the same fragment coding for DsrA, gave highest scores and lowest
e-values for DsrN of SAG-C11 in BlastX searches. In addition, DsrN sequences formed a stable
cluster with DsrN of SAG-C11 in phylogenetic analysis together with sequences of the phylum
Firmicutes but not with DsrN homologous cobyrinic acid a,c diamid synthase sequences of the
phylum Chloroflexi using the Neighbor Joining algorithm (Figure 3-20). This was confirmed by using
the Maximum Likelihood or Minimum Evolution algorithm (data not shown). Sequence similarities of
DsrN amino acid sequences ranged from 50% to 95% when compared to DsrN of Dehalococcoidia
SAG-C11 in global alignments using sequences of 58 to 485 amino acids (see Supplementary Table
7-18), corresponding nucleotide sequence similarities to dsrN of SAG-C11 ranged from 59% and
96%. BlastX analysis of the dsrA sequence derived from tidal flat sediments of the Wadden Sea gave
highest scores and lowest e-values for DsrA of SAG-C11. The DsrA and the DsrN sequences affiliated
with the respective genes of SAG-C11 in phylogenetic analysis (Figure 3-19 and Figure 3-20). The
285 amino acid DsrA sequence showed 100% similarity with DsrA of SAG-C11 in global alignments
and the respective nucleotide sequence 98%. The DsrN amino acid and nucleotide sequences both
75
Results
gave 97% similarity to DsrN and dsrN of SAG-C11, respectively. DsrA sequences derived from
Baffin Bay sediments gave highest scores and lowest e-values for DsrA of SAG-C11 or uncultured
sulphate reducer (Table 7-18).
Branch colours Figure 3-19: Phylogenetic tree based on a global alignment of DsrA sequences of ~259 amino acids, using the
Neighbor-joining method implemented in Mega 6.0 with 1000 bootstrap tests and pairwise deletion, evolutionary
distance were computed with the p-distance method. Sequences amplified in the current study are marked with a
green caption. DsrA of SAG-C11 is highlighted with a green background color. Numbers show bootstrap values
above 0.5.
76
Results
Amino acid sequences similarities of DsrA sequences derived from sediments of the Baffin Bay
were between 81% to 85% to DsrA of Dehalococcoidia SAG-C11 in global alignments and
corresponding nucleotide sequence similarities ranged from 74% to 78%. Three of the obtained DsrA
and DsrB sequences clustered with DsrA or DsrB sequences of SAG-C11 in phylogenetic analysis
(Figure 3-19 and Figure 3-18).
Figure 3-20: Phylogenetic tree based on DsrN sequences and DsrN homologs (cobyrinic acid a,c diamid
synthase) of ~450 amino acids using the Neighbor joining method implemented in Mega 6.0 with 1000 bootstrap
tests and pairwise deletion, evolutionary distance were computed with the p-distance method. Sequences
amplified in the current study are marked with green captions. Green branches indicate the ‘SAG-C11-DsrN
cluster’. DsrN or cobyrinic acid a,c diamid synthase sequences of classified members of the phylum Chloroflexi
are highlighted in green. Numbers show bootstrap values above 0.5.
BlastX analysis of dsrN sequences gave highest scores and lowest e-values for DsrN of
Dehalococcoidia-C11, and clustered with DsrN of SAG-C11 suggesting that the dsr sequences most
likely belong to Chloroflexi. Two of the DsrA sequences obtained from sediments of the Baffin Bay
affiliated with sequences of the ‘uncultured DsrAB lineage 2’ (201). Their respective DsrN sequences
affiliated with DsrN of SAG-C11 which clustered with DsrN sequences of members of the Firmicutes
77
Results
or DsrN homologous cobyrinic acid a,c diamid synthase sequences of other Chloroflexi strains
including strains of the class Dehalococcoidia (Figure 3-20).
3.6.3.1 Comparison of the nucleotide sequence similarity of sequences obtained from
amplifications with dsr nucleotide sequences of SAG-C11 and of Caldiserica
The 2.5 kb inserts of the three clones AA011, AA020 and AA075 were sequenced using internal
primers (Table 2-20). The gene sequence was luxR-dsrA-dsrB as observed in Dehalococcoidia SAGC11 (Figure 3-16). Alignments of the three fragments amplified from sediments revealed 98–100%
nucleotide sequence similarities with Dehalococcoidia SAG-C11 after global alignments. The DsrA
sequences gave highest BlastX scores and lowest e-values for DsrA of Dehalococcoidia SAG-C11.
DsrA sequences clustered similar as observed for the respective dsrB and dsrN sequences with dsr
gene sequences of SAG-C11 (Figure 3-19).
Six clones containing a 3.9 kb fragment were sequenced by gene walking using internal primers.
All fragments showed the same dsr gene organization as observed in SAG-C11: dsrA-dsrB-dsrDdsrN-dsrC (Figure 3-16). The same dsr operon organization was observed in Caldiserica sp.
(AQSQ01000030) (202). In addition, DsrA and DsrB sequences of Caldiserica closely affiliated with
DsrAB sequences of genes amplified in the current study and SAG-C11. Therefore, the dsrABDN gene
sequences derived from marine sediments of the Bay of Aarhus in the current study were compared
with the dsrABDN gene sequence of SAG-C11 and the dsrABDN gene sequence of Caldiserica in
global alignments using the p-distance method implement in Mega 6.0 (Table 3-6). Sequences
amplified from sediments had 66% to 97% nucleotide sequence similarities with the dsrABDN
sequence of SAG-C11 and 60% to 62% nucleotide sequence similarity with the dsrABDN sequence of
Caldiserica. All analysed sequences showed higher overall similarities to the dsrABDN nucleotide
sequence of SAG-C11 than to dsrABDN nucleotide sequence of Caldiserica.
Table 3-6: Nucleotide sequence similarities of selected clone inserts compared with dsr genes of
Dehalococcoidia SAG-C11 and dsr genes of Caldiserica.
clone
290
285
271
265
252
215
dsrABDN nucleotide sequence
similarities SAG-C11
74%
84%
66%
97%
68%
96%
dsrABDN nucleotide sequence
similarities Caldiserica
62%
62%
60%
61%
61%
61%
3.6.3.2 Inferring species level from dsrAB sequences
Selected dsrAB sequences amplified in the current study were compared with the dsrAB gene
sequence of Dehalococcoidia SAG-C11 to gain analyse phylogenetic diversity of dsrAB sequences
potentially linked to Chloroflexi (Table 3-7). The dsrAB sequences derived from primer binding
78
Results
position dsr1F and dsr4R have been used previously to assess species diversity by relating 16S rRNA
genes sequences diversity to dsrAB gene sequences diversity. For this purpose, an approximately 1.9
kb dsrAB gene stretch was used (150, 203). A nucleotide sequence similarity of 76% suggests another
genus level within the class Dehalococcoidia harbours genes for dissimilatory sulphite reduction.
An additional marker is the average GC content of the dsrAB sequence compared to the genome.
The SAG-C11 GC content is 47.8% and its respective dsrAB sequence 48.6%. All analysed dsrAB
sequences amplified in the current study showed a similar GC content as the genome of SAG-C11.
Table 3-7: Comparison of selected 1761 nucleotide dsrAB sequences amplified in the current study with primers
modified according to primers dsr1F und dsr4R with the dsrAB sequence of SAG-C11. Similarities were
calculated with p-distance method, pairwise deletion and 1000 bootstraps.
Clone
Aarhus_290
Aarhus_285
Aarhus_271
Aarhus_265
Aarhus_252
Aarhus_215
Aarhus_075
Aarhus_020
Aarhus_011
BaffinBay_048
BaffinBay_038
BaffinBay_022
BaffinBay_014
dsrAB nucleotide sequence similarity to C11
75.7%
86.5%
70.1%
97%
71%
96.9%
98.3%
98.9%
99.9%
75.8%
72.8%
75.8%
72.6%
79
GC content
48.6%
49.7%
48.1%
48.6%
47.5%
48.4%
48.7%
48.6%
48.6%
49.2%
47.6%
49.0%
47.6%
Discussion
4
Discussion
Dehalococcoidia belong to one of the most widespread and abundant bacterial groups in anoxic
marine sediments (34, 40-51, 204). All cultivated and isolated members of the class Dehalococcoidia
depend on organohalide respiration. However, recent studies of single amplified Dehalococcoidia
genomes and pan genomes provided evidence that the class Dehalococcoidia is more versatile and that
not all members depend on reductive dehalogenation (46, 48, 62). Nevertheless, the detection of 16S
rRNA
and
rdhA
gene
sequences
in
marine
sediments
affiliating
with
sequences
of
organohalide-respiring Dehalococcoidia, suggest that Dehalococcoidia subgroups depending on
organohalide respiration occur in marine sediments. Most studies conducted with cultivated
Dehalococcoidia focused on the investigation of dehalogenation of xenobiotics. It thus remained
poorly understood which natural halogenated electron acceptors could allow for growth and energy
conservation in marine non-contaminated sediments. In addition, different dehalogenation patterns and
substrate spectra were observed for organohalide-respiring Dehalococcoidia but little was known
about chemical properties of an electron acceptor onto reductive dehalogenation. D. mccartyi strain
CBDB1 was used as a model organism and cultivated with different halogenated electron acceptors to
gain a deeper understanding of chemical parameters required of a compound to serve as electron
acceptor for organohalide respiration by Dehalococcoidia. The tested compounds contained different
halogens, substituents and heteroatoms. This may allow for the identification of potential natural
electron acceptors of Dehalococcoidia and a further insight into the role of organohalide respiring
Dehalococcoidia in transformation of diverse halogenated xenobiotics. A special focus was placed on
brominated aromatic compounds, since they have been reported to occur naturally in marine
environments (128, 132, 133, 136-139), but are also introduced into the environment from
anthropogenic sources, i.e. as flame retardants (66, 191). Additional to chemical parameters the role of
enzymatic properties of a strain for its dehalogenation spectra and patterns was investigated for
brominated benzenes using activity assays and shotgun proteomics. Molecular methods were applied
to investigate potential electron acceptors of so far uncultivated and non-dehalogenating members of
the class Dehalococcoidia in marine sediments. Together this research contributes to the further
understanding of the respiratory modes of Dehalococcoidia and the reasons for their widespread
distribution and abundance in marine sediments. Additionally, this research provides insights into the
mechanisms of reductive dehalogenation and may contribute to an improved fate prediction of
halogenated compounds from natural and anthropogenic sources in the environment.
80
Discussion
4.1
Strategies to investigate electron acceptors used by bacteria of the class
Dehalococcoidia
Different strategies were developed and applied in the current work to gain insight into the
electron acceptor diversity of Dehalococcoidia which may elucidate why members of the class
Dehalococcoidia occur abundantly in marine non-contaminated sediment sites. These strategies
included i) the cultivation of Dehalococcoides mccartyi model strain CBDB1 ii) resting cell activity
assays to screen and measure transformation rates of electron acceptors of organohalide-respiring
Dehalococcoidia and marine dehalogenating microcosms iii) the analysis of reductive dehalogenase
expression in D. mccartyi model strain CBDB1 after cultivation with different electron acceptors iv)
the characterization of chemical properties of electron acceptors tested with model strain CBDB1, v)
the analysis of the diversity and distribution of functional genes in Dehalococcoidia associated with
respiratory functions via molecular tools. These strategies are discussed briefly in this chapter while
the results from the different strategies will be discussed in later chapters.
4.1.1
Cultivation of the model organism D. mccartyi strain CBDB1
One strategy selected was the cultivation of D. mccartyi strain CBDB1 with different potential
electron acceptors as model organism for Dehalococcoidia. In marine sediments 16S rRNA gene
sequences were detected affiliating with cultivated D. mccartyi strains (40). Additionally, reductive
dehalogenase homologues genes that were highly similar to reductive dehalogenase sequences from
strain CBDB1 were reported in marine sediments (60, 61). These findings suggested the presence of
organohalide-respiring Dehalococcoidia in marine non-contaminated sediments related to cultivated
D. mccartyi strains such as strain CBDB1 isolated from terrestrial sites. The usage of strain CBDB1 as
model organism allowed for the investigation of growth and transformation of halogenated electron
acceptors in fully synthetic medium, under defined conditions as described previously for D. mccartyi
strains and various chlorinated electron acceptors (7-16). Attempts for enrichment and cultivation of
Dehalococcoidia from marine sites were conducted in a parallel project by Camelia Algora. Also in
the current study, cultivation attempts of marine Dehalococcoidia were carried out but remained
unsuccessful (data not shown). Previous studies focused on the transformation of halogenated
contaminants from anthropogenic sources by D. mccartyi strains (10, 12, 104, 118, 205), in the current
study the electron acceptors for cultivation were however chosen based on their functional groups and
halogen substituents and not primarily based on their potential as contaminant. This allowed for an
investigation of the basic parameters that determine whether a compound is used as an electron
acceptor by a D. mccartyi strain or not. Results may allow for an extrapolation onto other
organohalide-respiring Dehalococcoidia and naturally occurring halogenated compounds (131, 139,
206) to estimate their potential to promote growth of organohalide-respiring Dehalococcoidia in
81
Discussion
marine sediments and eventually may allow for cultivation and enrichment of Dehalococcoidia from
marine sediments.
4.1.2
Activity measurements in a microtiter plate-based assay for identification of new
halogenated electron acceptors
D. mccartyi strain CBDB1 was also used as a model organism to establish a resting-cell activity
assay in a 96-well microtiter plate format, enabling fast screening of halogenated compounds towards
their use as electron acceptor by organohalide-respiring bacteria, including organohalide-respiring
Dehalococcoidia. Reductive dehalogenase activity assays using viologens as artificial electron donor
were shown to be a sensitive tool to investigate the presence of reductive dehalogenases independent
of molecular tools and have been used previously to investigate activity with different electron
acceptors of D. mccartyi strains (95, 105, 111, 190). In the current study resting cells of D. mccartyi
strain CBDB1 were used as catalysts to measure the transformation rate of halogenated compounds
while other studies used a similar assay to characterize crude extracts of D. mccartyi cells or purified
reductive dehalogenases (95, 105, 111, 190). The assay is believed to operate with whole cells because
the catalysing enzymes are located on the outer side of the outer membrane and are therefore
accessible by viologens as electron donor and halogenated electron acceptors (29). The 96-well
microtiter plate format assay established in this study allowed for quick screening of multiple electron
acceptors in parallel, including several control set-ups, while using only small amounts of resting cell
suspension. The evaluation of the activity assay with a photometer in comparison with the evaluation
by chromatography-dependent methods did not require establishing of different analytical methods for
each investigated electron acceptor and allowed testing of undefined and/or complex molecules such
as for instance humic acids. Additionally, photometry allowed monitoring of reductive dehalogenation
in real-time (94). It allowed therefore for a quick screening and rate determination of electron
acceptors for organohalide-respiring bacteria.
Parallel activity assays, evaluated by analysis of reaction products with GC-FID, yielded similar
results when using the same culture and electron acceptors for comparison, demonstrating that the 96well microtiter plate-based activity assay is suitable for the screening of new electron acceptors.
However, photometer and GC-FID evaluated activity assays only assess the activity of already
expressed proteins, not of those that are encoded in the genome but which are not expressed under the
cultivation conditions used for the preparation of resting cells. This might lead to differences in the
results obtained from cultivation and in vitro activity tests, which was observed for several compounds
despite their transformation in culture by strain CBDB1. Such results could indicate that specific
reductive dehalogenases were not expressed under the cultivation conditions used for preparation of
the resting cells for the activity assay. However, when cells were previously cultivated with the same
electron acceptor which was subsequently used in activity assays, no improved activity rates for
electron acceptors tested was observed suggesting, the absence of measurable activity was not related
82
Discussion
to reductive dehalogenase expression at least for those compounds tested in the current study (see also
4.2.3). Instead, the failure to detect activity in resting cell activity assays with monobromobenzene,
2-chloro-6-fluorobenzonitrile or 4-bromo-3,5-dihydroxybenzoic acid, despite observed activity in
cultures, might have been rather related to the lower sensitivity of the activity test compared to
cultivation. Attempts to increase the sensitivity of the activity test by using a higher amount of cells,
an electron donor with lower redox potential such as ethyl viologen (190, 207) or increasing the
incubation time, did not allow detection of activity. This suggests that the test in its current form does
not work with certain reductive dehalogenases. This could be due to incompatibility of the used
electron donor with the dehalogenase, pH, ionic strength, temperature or other conditions. Some
reactions might be extremely slow and not suitable for in vitro assays.
In the microtiter plate assay a background decolourization, i.e. slow and continuous oxidation of
the reduced methyl viologen, was measured in the presence of resting cells, even if no halogenated
electron acceptor was added. Inhibition studies conducted with alkyl iodides specifically inhibiting
cobalamin-dependent enzymes (94, 111) did not show a decrease of background methyl viologen
oxidation, indicating that reductive dehalogenases or other corrinoid-containing enzymes were not
responsible for this slow oxidation of methyl viologen (Figure 3-8). Medium components such as
vitamins contributed only to a low amount to the measured background oxidation of methyl viologen.
Tests with cells exposed to more than 80°C did not show background oxidation of methyl viologen
indicating that heat sensitive cell components induced the background methyl viologen oxidation.
Together the data suggested that a protein was responsible for oxidation of methyl viologen. A
possible reason for the observed background activity is the presence of hydrogenase activity. Protons
could serve as alternative electron sink for electrons provided by reduced methyl viologen at low
redox potential (standard redox potential MV2+/MV-+ -446 mV) (207, 208). The transfer of electrons
by methyl viologen and hydrogenases could drive the production of hydrogen (ΔEo' H+/H2 -414 mV)
as it was described in previous studies (209-212). Findings from literature about reactions that could
participate in methyl viologen reactions together with the conducted experiments in the current study
suggested that reductive dehalogenases were not involved in the transfer of electrons to a so far
unknown electron acceptor. Therefore, the background activity measured in negative controls was
subtracted from samples containing organohalogens to obtain the reductive dehalogenase activity and
transformation rates of halogenated compounds by resting cells used as catalyst.
The established 96-well microtiter plate activity assay was used to investigate new electron
acceptors of D. mccartyi strain CBDB1. The transformation of halogenated compounds in culture with
D. mccartyi strain CBDB1 suggests that a variety of halogenated compounds can principally serve as
electron acceptor for organohalide-respiring Dehalococcoidia, the reaction itself also depends on the
presence of appropriate enzymes in the studied environment. Tidal flat sediments, marine sediments
and marine deep-sea sediments (depths greater than 500 m) and dehalogenating sediment microcosms
were studied for reductive dehalogenation activity using the 96-well microtiter plate-based assay or
83
Discussion
the GC-FID-evaluated assay but no activity was detected. For a successful detection of dehalogenation
activity, the encoding genes must be present in the sediments but additionally the enzymes must be
expressed. The failure to detect dehalogenation activity directly in sediments underscores the need for
previous enrichment by cultivation. However, while cultivation and enrichment with halogenated
electron acceptors allows for a more sensitive detection of potential reductive dehalogenation activity
in marine sediments, enzymes might be induced during cultivation which were not expressed under
the natural conditions in the sediment environment. Possibly, a higher biomass of cells directly
extracted from freshly sampled sediments will allow in the future to use the activity assay as a strategy
to detect dehalogenation activity in crude extracts of sediments without prior enrichment with
halogenated compounds by cultivation.
4.1.3
Analysis of reductive dehalogenase expression as a strategy to gain insights into
enzyme specificity
Cultivated species of the class Dehalococcoidia typically contain several reductive dehalogenase
homologues (29) but only for few reductive dehalogenases it is known which electron acceptors
induce their transcription (103, 106, 107). Therefore, also electron acceptors, which may induce
reductive dehalogenases of marine organohalide-respiring microorganisms, remain unknown. Shotgun
proteomics of proteins extracted from marine sediments could be used to investigate natural reductive
dehalogenation activity in marine sediments. However, metagenomic data for comparison would be
required and the separation of sufficient amounts of proteins in high quality for proteomic
measurements seems unrealistic at present for sediments in which dehalogenation activity cannot be
detected by activity assays. Therefore, in the current study reductive dehalogenase activity after
induction with specific electron acceptors was analysed using activity assays with resting cells of the
model organism strain CBDB1. Additionally, shotgun proteomics with cells of strain CBDB1 grown
with different electron acceptors was used to gain insights into potential functions of the diverse
reductive dehalogenases in members of the class Dehalococcoidia. Using shotgun proteomics for a
prediction of potential functions was used previously for an uncharacterized Dehalococcoidescontaining mixed culture (106). In the current study shotgun proteomics and activity assays were
mainly used to understand the importance of different halogen types onto the expression of reductive
dehalogenases using different bromobenzene congeners. This contributed to the understanding of
potential electron acceptors of reductive dehalogenases in marine sediments (see also chapter 4.2.3,
page 93).
84
Discussion
4.1.4
Evaluation of density functional theory-based data as a strategy to elucidate chemical
parameters influencing reductive dehalogenation
Thousands of halogenated molecules occur in the environment (125, 139, 206). However, due to
their diversity and often low concentration as individual compounds, testing of the multitude of
detected compounds with organohalide-respiring Dehalococcoidia for reductive dehalogenation by
cultivation or activity assays is difficult: Often, identified natural compounds are commercially not
available and their isolation from natural environments in sufficient amounts for cultivation or activity
assays is challenging. The identification of chemical properties, which influence reductive
dehalogenation, could allow for a classification of halogenated compounds with respect to their
potential to serve as electron acceptor for organohalide-respiring Dehalococcoidia independent of
cultivation or activity assays (see also 4.3). This could contribute to a deeper understanding of the
ecological niche of Dehalococcoidia and a better fate prediction of halogenated compounds from
natural or/and anthropogenic sources in the environment. In silica modelling of a molecule’s chemical
properties could provide such a tool. For this, chemical parameters of halogenated electron acceptors
were modelled using density functional theory, a method previously described to model electronic
structures (213). Several parameters were evaluated for their ability to predict transformation
pathways catalysed by reductive dehalogenases of strain CBDB1. Partial charges revealed to serve
best as a predictor of dehalogenation pathways which is in agreement with previous studies using
Mulliken partial charges to predict dehalogenation pathways of chlorobenzenes, dioxins and
chlorophenols catalysed by strain CBDB1 (195). In the current study, the Mulliken model was
extended to a more diverse set of electron acceptors, including molecules with diverse functional
groups and different types of halogens occurring in natural halogenated electron acceptors. By
extending the Mulliken model to a broader set of halogenated electron acceptors, several molecules
were identified whose transformation pathways were not predicted correctly. The Mulliken model is
the oldest method for partial charge modelling and was described to have a strong dependence on the
basis sets (187, 214). Moreover, the Mulliken model does not take into account differences in
electronegativity of different atoms, which can be especially important when comparing different
halogen substituents with different electronegativity (215). Therefore, in the current study, the
Hirshfeld (188) and the NBO (197) partial charge models were added for the evaluation of
transformation pathways. The Weinhold-Reed Natural Population Analysis to obtain natural bond
orbital (NBO) partial charges is considered as an improved procedure compared to the Mulliken
analysis (196, 214). Both models depend on basis functions that represent wave functions (214). The
Hirshfeld model is less basis set-dependent and can be calculated directly via density functional theory
using a hypothetical promolecule (188, 215). By using several partial charge models, a verification of
observed trends was achieved, while minimizing potential errors. All tested models indicated that the
observed regioselective removal of halogens is governed by the most positive halogen partial charge
85
Discussion
with up to 96% correct prediction rates for the transformation pathways of strain CBDB1. However,
each model showed inconsistency with specific molecule types which could be related to the different
approaches used for calculating the charge distribution i.e. for Hirshfeld the portioning of electron
densities and the use of a promolecule and for Mulliken and NBO the analysis of wave functions
(214). For instance, modelling of partial charges revealed that not all partial charge models are suitable
to predict the dehalogenation pathways compounds with both, bromine and chlorine substituents.
Mulliken and Hirshfeld charges calculated with the Gaussian 09 revision C.0149 program and density
functional theory level B3LYP/6-31G(d,p) yielded generally more positive partial charges for chlorine
substituents than for bromine substituents when comparing brominated and chlorinated congeners.
Both models assigned to bromine atoms a more negative charge than to chlorine atoms, thus not
corresponding to a higher electronegativity of chlorine compared to bromine (216). Although
Mulliken and Hirshfeld charges predicted correctly the transformation pathway when the molecule
was substituted with only one type of halogen, both models did not predict correctly a more extensive
dehalogenation of brominated benzenes or phenols than of chlorinated benzenes or phenols when
using the most positive halogen charge as predictor for the removal of halogens. This trend was
reflected in the prediction of the dehalogenation of 2-chloro-3-bromopyridine to 2-chloropyridine.
Only the NBO model was suitable for a comparison of electron acceptors substituted with both
bromine and chlorine.
The variance found in the different models emphasizes the need for several charge models applied
in parallel to allow for a verification of obtained values. Additionally, the influence of the basis-sets
used for calculation of the partial charge will need to be investigated, however, this was beyond the
scope of the current study and will need to be investigated in future studies. Also with the current
approach, it was not possible to define an absolute threshold for the positive charge on a halogen
substituent to predict dehalogenation. For this, an even broader data set of different electron acceptors
might have to be evaluated. However, the usage of modelled chemical descriptors allowed for the
description of chemical properties which influence reductive dehalogenation catalysed by strain
CBDB1 beyond classical chemical concepts such as inductive and mesomeric effects (see for further
discussion section 4.3). In addition, this approach may harbour the potential to elucidate chemical
properties influencing reductive dehalogenation of different organohalide-respiring bacterial strains,
possibly also from marine sites.
4.1.5
Analysis of Dehalococcoidia genes associated with respiratory functions in marine
sediments
Another strategy to investigate potential electron acceptors of marine Dehalococcoidia is the
analysis of the presence of functional genes associated with respiratory functions in Dehalococcoidia.
This analysis was only possible due to the recent development of single cell genome sequencing
technologies because only these allowed the design of specific primers and a correlation of functional
86
Discussion
genes with phylogenetic marker genes. The information retrieved from the genomic content of
Dehalococcoidia single-cells was used as a basis in the current study to investigate the potential of
Dehalococcoidia to use non-halogenated electron acceptors. A gene with highest similarities to a
reductive dehalogenase subunit A was identified in one of the single cells (personal communication
Dr. Kenneth Wasmund, UFZ, Leipzig) however it was not associated with a TAT leader sequence or a
gene coding for the membrane anchor RdhB. Therefore, its function in a respiratory process as known
from other organohalide-respiring Dehalococcoidia strains seemed unlikely. Instead, several other
genes associated with respiratory functions were identified in the Dehalococcoidia genomes.
Dissimilatory sulphite reduction was described so far only in a restricted number of bacterial phyla
and many dsr sequences remain with unknown phylogeny. Therefore, dsr genes were selected for
further investigation in the current study by a targeted amplification of dsr genes by PCR because it
provides insight into the reasons for Dehalococcoidia diversity in marine sediments but also into the
general ecology of marine sediments. As a source DNA had to be isolated from marine sediments in
sufficient quality to allow for the amplification of functional genes. In this study fragments of up to
3.9 kb were amplified to allow for a detailed analysis of phylogeny and gene order of the amplified
sequences. Methods had to be chosen which avoided the sheering of DNA into small fragments. For
the isolation of DNA from marine sediments different methods were tested which ranged from
commercially available purification kits to methods using density centrifugation to isolate cells from
sediments and subsequent DNA extraction procedures as were described previously (166, 167, 217).
While DNA purification kits have the advantage of a standardized protocol they have the disadvantage
that only 0.5 g of sediment can be applied to the extraction procedure or many extractions have to be
done in parallel. Density centrifugation with Nycodenz was used in the current study beside
commercially available kits, as it allowed for the extraction of cells from larger amounts of sediments,
which was important especially for sediments with lower cell numbers and decreasing amounts of
extractable DNA. However, Holmsgaard and colleagues showed that microbial populations extracted
from soil using density centrifugation with Nycodenz had significantly lower biodiversity than the
microbial populations in the soil obtained with direct DNA extraction methods (217) suggesting that
also in the current study an important fraction of diversity within functional genes of Dehalococcoidia
might have been overlooked. Indeed, only few sequences coding for the targeted genes were obtained
after screening of hundreds of clones with a correct insert size (see for further discussion 4.3.6),
however, this approach allowed first insights into the functional gene diversity of the nondehalogenating community of the class Dehalococcoidia.
87
Discussion
4.2
The electron acceptor diversity of D. mccartyi strain CBDB1 and marine
dehalogenating microorganisms
In the current study, reductive dehalogenation of brominated aromatic compounds that resemble
naturally occurring brominated compounds (132, 135, 218) was investigated with strain CBDB1 and
marine dehalogenating consortia. In addition, several chlorinated and one fluorinated aromatic
compound with different functional groups have been tested with strain CBDB1 to account for the
structural diversity of natural halogenated compounds and halogenated contaminants from
anthropogenic sources and to further elucidate the role of Dehalococcoidia and marine dehalogenating
microorganisms in the fate prediction of halogenated compounds in anaerobic sediment environments.
Dehalococcoidia model organism D. mccartyi strain CBDB1 dehalogenated brominated benzenes,
phenols, pyridines, furoic acids and benzoic acids and was able to grow during their dehalogenation.
Strain CBDB1 furthermore dehalogenated chlorinated anilines, thiophenes and benzonitriles in
cultivation and/or in activity assays. These findings demonstrate the diversity of electron acceptors
which potentially can support growth of organohalide-respiring Dehalococcoidia in sediment
environments and could explain the occurrence of reductive dehalogenase homologous and 16S rRNA
gene sequences related with organohalide-respiring representatives of the class Dehalococcoidia in
non-contaminated sediment sites. Reductive dehalogenation of brominated aromatic compounds was
furthermore observed when testing marine microcosms suggesting that the bacterial community in
marine sediments participates in the natural halogen cycle and the transformation of halogenated
xenobiotics in the environment.
4.2.1
Growth of D. mccartyi strain CBDB1 with halogenated electron acceptors
Strain CBDB1 was cultivated with different halogenated aromatic compounds to study the
electron acceptors diversity that may sustain growth of organohalide-respiring Dehalococcoidia.
Growth of strain CBDB1 was shown with 2,3-dichloroanilines and 2-chloro-6-fluorobenzonitrile and
brominated phenols, brominated furoic acids and brominated benzoic acids. Several of the tested
halogenated compounds, including two dichloroanilines and both tested dichloropyridines did not
allow for growth of strain CBDB1, demonstrating that functional groups, the type and the degree of
halogenation influence reductive dehalogenation and thereby growth of D. mccartyi strain CBDB1.
Obtained growth yields of strain CBDB1 cultivated with 2,3-dichloroaniline and 2-chloro-6fluorobenzonitrile are well in agreement with growth yields obtained in previous studies with strain
CBDB1 cultivated with chlorinated phenols, ethenes and benzenes (7, 10, 11, 119). No formation of
2-chlorobenzonitrile was observed and 2-fluorobenzonitrile was not transformed by strain CBDB1
suggesting that growth of strain CBDB1 with 2-chloro-6-fluorobenzonitrile was supported by
dechlorination and not by defluorination. Bacterial growth via defluorination was demonstrated with
monofluoroacetate (219). However, monofluoroacetate served as carbon source and not as respiratory
88
Discussion
electron acceptor for the aerobic Moraxella strain (220). Growth with chlorinated anilines was
reported for instance with 4- and 3-chloroaniline when used as a carbon sources (221, 222).
All brominated compounds tested in the current study allowed for growth of strain CBDB1.
Brominated compounds were reported to be especially abundant in marine environments (139) but
little information about growth of cultivated members of the Dehalococcoidia with brominated
aromatics was so far available. Previous studies observed growth of D. mccartyi strain BAV1 with the
aliphatic vinyl bromide (10) however, growth yields were not reported. For a D. mccartyi- and
Desulfovibrio-containing co-culture grown with the aromatic polybrominated diphenylether as
electron acceptor, growth yields of 2 x 1014 cells mol-1 halogen released were estimated for the
D. mccartyi
sub-population
(15).
In
comparison
to
that,
growth
yields
ranging
from
0.1 x 1014 to 1.8 x 1014 cells mol-1 halogen released were obtained in the current work for strain
CBDB1 cultivated with brominated electron acceptors. Growth yields were lower with compounds
that either reacted abiotically and/or contained phenolic functional groups such as phenols or
hydroxybenzoic acid than with compounds which were stable or which contained no hydroxy
functional groups (Figure 4-1). Possibly, the biotically-catalysed release of bromide was overestimated
for cultures containing strain CBDB1 or strain CBDB1 did not completely couple the biotic release of
bromine to growth. Brominated electron acceptors containing hydroxy groups showed lower growth
yields compared to electron acceptors containing methoxy or carboxy groups. For instance 4-bromo3,5-dihydroxybenzoic acid yielded 0.4 x 1014 cells mol-1 halogen released while 4-bromo-3,5dimethoxybenzoic acid yielded 1.8 x 1014 cells mol-1 halogen released suggesting inhibitory effects of
the phenolic compounds onto growth of strain CBDB1. Moreover, growth of strain CBDB1 with
bromophenols was achieved only after initial amendment of low dosages of bromophenols and a
sequential addition of increasing doses of bromophenols along with continuing growth of strain
CBDB1 (see section 3.1.5). A similar effect was observed during cultivation of strain CBDB1 with
chlorinated phenols (119). Boyle and colleagues reported lower growth yields of Desulfovibrio strain
TBP-1 when grown with 2,4,6-tribromophenol compared to sulphate as terminal electron acceptor in
spite of sulphate being the energetically less favourable electron acceptor (141). Phenols were
described previously to interfere with the membrane proton gradient and the generation of ATP (223225) and pentachlorophenol was used as respiratory inhibitor (226). Toxic or inhibitory effects of the
hydroxy group may therefore explain lower growth yields of strain CBDB1 (Figure 4-1). Another
possibility is that different electron acceptors allow for different extent of proton translocation, which
needs to be studied further by biochemical methods.
Many reported brominated compounds from marine environments produced by algae,
hemichordates and sponges as chemical defence mechanisms against bacterial growth contain
phenolic groups and have been shown to inhibit microbial activity (227, 228) and could therefore
counteract growth of Dehalococcoidia in marine sediments. Interestingly, in Dehalococcoidia pangenomes RBG-1351 and RBG-2, the genetic potential to degrade hydroxylated aromatics via
89
-1
growth yield (cells mol halogen)
Discussion
6e+14
5e+14
4e+14
3e+14
2e+14
1e+14
2,3-D
2,3-D CA (I)
CA
2,4-D (II)
C
2,6-D A
2,3-D CA
C
2,5-D BN
C
2,6-D BN
2-C-6 CBN
2,4,6- FBN
TBPh
2,4-D
BPh
4-B-3 2,6-DBPh
,5
4-B-3 -DMBA
,5-DH
2,3-D BA
C
2,5-D Th
4,5-D CTh
B-25-B-2 FA
2,3-D FA
CPy
2,6-D
3-B-2 CPy
-CPy
PCE
TCE
2,3-D
CPh
MBB
1,2-D
B
1,3-D B
BB
1,4-D
1,2,4- BB
TBB
HCB
VC_B
TCE_ av1
FL
VC_G2
T
0
electron acceptor
Figure 4-1: Comparison of growth yields obtained with strain CBDB1 (all values but the last three), BAV1, FL2
and GT (indicated in the sample name) cultivated with different halogenated electron acceptors.
pyrogallol hydroxytransferases were detected (144). In DEH-SAG-C11 several genes for degradation
of phenolic compounds have been detected (personal communication, Dr. Kenneth Wasmund, UFZ,
Leipzig). Therefore, phenolic compounds could serve also as carbon source for several
Dehalococcoidia subgroups. Generally, brominated compounds were shown in this study to support
growth of D. mccartyi strain CBDB1 even in presence of phenolic groups and could therefore
potentially sustain growth of organohalide-respiring Dehalococcoidia in marine sediments. In
addition, for several brominated compounds growth yields similar to those observed for D. mccartyi
strain CBDB1 cultivated with chlorinated phenols (119), tetra- and trichloroethene (194) and for
D. mccartyi strains BAV1, FL2 and GT, grown with different chlorinated ethenes (10-12) were
obtained indicating that brominated electron acceptors are equally well suited as chlorinated electron
acceptors to support growth of organohalide-respiring Dehalococcoidia (Figure 4-1). The ability of
D. mccartyi strains to grow with a broad variety of brominated and chlorinated electron acceptors
underscores the important role of Dehalococcoidia species in the natural halogen cycle and in fate
prediction of halogenated compounds in the environment.
4.2.2
Dehalogenation pathways of halogenated electron acceptors catalysed by strain
CBDB1
Dehalogenation pathways catalysed by strain CBDB1 were investigated by cultivation and/or by
GC-FID evaluated activity assays. Results of the current study demonstrated that dehalogenation
pathways catalysed by strain CBDB1 follow distinct rules similar as previously reported for
90
Discussion
chlorinated benzenes, phenols and dioxins (7, 119, 121). However, results of the current study
demonstrated that these rules become are complex for electron acceptors containing different halogen
types and functional groups.
Halogens of chlorinated benzenes have been previously classified as doubly flanked, singly
flanked or isolated when they have two, one or no neighbouring chlorine substituent, respectively
(122). In the current study, these terms are extended as follows: halogen atoms flanked by
hydrogen-substituted carbon atoms in both ortho positions to the halogen are referred to as isolated.
Halogens flanked in ortho position by a hydrogen-substituted carbon and one functional group
different to a hydrogen-substituted carbon including heteroatoms, are referred to as singly flanked.
Halogen substituents flanked in both ortho positions by a functional group different to a
hydrogen-substituted carbon are referred to as doubly-flanked.
The removal of isolated chlorine substituents by strain CBDB1 was not observed in the current
study. This is in agreement with previous studies using chlorinated phenols, benzenes, biphenyls or
dioxins as electron acceptor for strain CBDB1 (7, 118, 119, 124). The removal of isolated chlorine
substituents was reported previously for aliphatic compounds and D. mccartyi species (8, 10, 12), but
not for aromatic compounds so far. However, in mixed cultures also the dehalogenation of isolated
substituents was shown but it remains to be elucidated whether this process is catalysed by reductive
dehalogenases and in metabolic or co-metabolic processes (229, 230).
Singly flanked chlorines were dehalogenated for some but not all of the compounds tested. For
2,6-dichlorobenzonitrile, for instance, the singly flanked chlorine was dehalogenated whereas the
singly flanked chlorine of 2,6-dichloroanilines were not removed indicating an enhancing effect of the
cyano group onto reductive dehalogenation. In addition, the singly flanked fluorine of 2-chloro-6fluorobenzonitrile was not removed whereas the singly flanked chlorine atom was dehalogenated
indicating a preference of chlorine over fluorine during reductive dehalogenation by strain CBDB1. A
preferential dehalogenation of doubly flanked chlorines over singly flanked chlorines was observed for
most of the tested compounds including for instance the removal of the doubly flanked halogen of 2,3dichloroaniline and -benzonitrile (199). Dehalogenation of anilines was reported in methanogenic
groundwater slurries (231) and for chlorinated benzonitriles in soil samples incubated under aerobic
conditions (232, 233). The removal of the doubly flanked chlorine of 2,3-dichloroaniline
or -benzonitrile in cultivation or activity assays is in agreement with previous studies showing strain
CBDB1 dehalogenated the doubly flanked halogens of 2,3-dichlorophenol or 1,2,3-trichlorobenzene
(7, 119). A preferential removal of singly flanked chlorine substituents compared to doubly flanked
chlorine substituents was observed for D. mccartyi strain 195 and 1,2,3-trichlorobenzene, however
only in presence of another halogenated electron acceptor (205).
However, exceptions of dehalogenation patterns of strain CBDB1 were observed. The doubly
flanked chlorine of 2,3-dichloropyridine or 3-bromo-2-chloropyridine was not removed. Instead, the
singly flanked bromine of 3-bromo-2-chloropyridine was dehalogenated (234) indicating a preferential
91
Discussion
dehalogenation of bromine over chlorine for reductive dehalogenation by strain CBDB1. In addition,
this demonstrates that a prediction of dehalogenation pathways based on dehalogenation patterns
observed with other molecule types is not sufficient but needs to be combined with chemical
properties of the electron acceptor.
When comparing dehalogenation pathways of brominated and chlorinated congeners in more
detail, similar dehalogenation pathways were observed, however, brominated electron acceptors were
always dehalogenated to a further extent than their chlorinated equivalents. The dehalogenation
pathway of 2,4,6-tribromophenol and 2,4-dibromophenol for instance were similar to those observed
for 2,4,6-trichlorophenol and 2,4-dichlorophenol (119). For all congeners the halogen substituents in
ortho-position to the hydroxy group were preferred over the isolated halogen substituent in paraposition. However, 2,4-dichlorophenol was converted only slowly and incompletely and 4chlorophenol was not dehalogenated (119) whereas 2,4-dibromophenol was fully dehalogenated to
phenol via 4-monobromophenol. A similar result was obtained for 1,2,4-tribromobenzene and 1,2,4trichlorobenzene. Both compounds were dehalogenated to the 1,3- and 1,4-dihalogenated benzenes (7,
193). However, while the isolated chlorine substituents of 1,3- and 1,4-dichlorobenzene were not
dehalogenated by strain CBDB1, the isolated bromine substituents of 1,3- and 1,4-dibromobenzene
were removed, yielding monobromobenzene and subsequently benzene (7, 193). Debromination was
reported for brominated biphenyl and D. mccartyi strains BAV1 and 195 (143). However,
debromination was incomplete and occurred only in presence of a chlorinated aliphatic compound.
The complete removal of all bromine substituents of an aromatic electron acceptor during reductive
dehalogenation was shown for polybrominated diphenylethers by a co-culture of D. mccartyi and
Desulfovibrio strains (15). However, a participation of the Desulfovibrio strain in debromination could
not be excluded since Desulfovibrio species were described to dehalogenate bromo- and iodophenols
(141, 142). For aliphatic compounds debromination of vinyl bromide by D. mccartyi strain BAV1 was
observed in culture, yielding ethene as non-halogenated end product (10) and by the purified TCE
reductive dehalogenase of D. mccartyi strain 195 dehalogenating various brominated alkanes and
alkenes.
Together, the more extensive dehalogenation of brominated compared to chlorinated aromatics by
strain CBDB1 indicates that organohalide-respiring Dehalococcoidia may have a more diverse range
of brominated electron acceptors than anticipated, especially for compounds with few or isolated
halogen substituents such as described to occur in marine sediments (see also Figure 1-2) (128, 131136, 139, 235). Findings of the current study suggest that organohalide-respiring Dehalococcoidia in
marine sediments may use brominated natural compounds with a low degree of halogenation as
electron acceptors for respiration. In addition, dehalogenation of compounds with a low degree of
halogenation as described for natural compounds has been shown in the current study to be facilitated
or inhibited by heteroatoms and functional groups also occurring in natural organohalogens (131).
92
Discussion
4.2.3
Specific activities of strain CBDB1 with halogenated electron acceptors
The rates at which halogenated compounds are transformed by D. mccartyi strain CBDB1 were
investigated in activity assays with resting cells, using methyl viologen as artificial electron donor.
Specific activities with brominated electron acceptors were lower than specific activities with their
chlorinated congeners. For instance, 1,2,4-tribromobenzene gave a specific activity of 25.8 ± 15.4 nkat
mg-1 protein, when previously cultivated with 1,2,4-tribromobenzene. Higher specific activities of
219.4 ± 110 nkat mg-1 protein were obtained when cells were previously cultivated with 1,2,3trichlorobenzene while the specific activity for 1,2,3-trichlorobenzene was 36 ± 9 demonstrating that
observed higher specific activities with 1,2-dibromo- and 1,2,4-tribromobenzene were not related with
the previous cultivation with a brominated congener. For 1,2,4-trichlorobenzene a specific activity of
only 0.3 nkat mg-1 protein was observed in a previous study (111). When comparing strain CBDB1
cells cultivated with the same electron acceptor, 1,2,4-tribromobenzene and 1,2-dibromobenzene
constantly gave higher specific activities than 1,2,3-trichlorobenzene and were dehalogenated to a
further extent than 1,2,3-trichlorobenzene. This is in agreement with previous studies using the
purified trichloroethene reductive dehalogenase of D. mccartyi strain 195 (95). 1,2-Dibromoethane
was reduced to ethene at a specific activity of 500 nkat mg-1 protein while 1,2-dichloroethane was
transformed at 125 nkat mg-1 protein. Vinyl bromide and vinyl chloride were transformed to ethene at
specific activities of 3 and 0.6 nkat mg-1, respectively (95). The finding that brominated electron
acceptors were transformed at higher rates compared to chlorinated electron acceptors in the current
study and in previous studies with brominated aliphatic electron acceptors (95) is in accordance with
observations of abiotic dehalogenation reactions, in which the aryl-bromine bond was shown to be
weaker than the aryl-chlorine bond (236, 237). In addition, when correlating specific activities of
brominated benzenes and phenols or chlorinated thiophenes and phenols with partial charges, more
positive partial charges of the halogen were paralleled with increased specific activity rates when
examining only one compound class. However, when the different compound classes were compared
no consistent trend was observed. Enhanced activity rates for compounds with more positive halogen
partial charges was observed for chlorobenzenes in previous studies (195). Enhanced dehalogenation
activity with brominated compounds compared to chlorinated compounds could be related with
different chemical properties of the halogen species such as electronegativity and size. Higher
transformation rates observed with brominated aromatic and aliphatic electron acceptors compared to
chlorinated compounds suggest that the ability to transform brominated electron acceptors could be
advantageous for Dehalococcoidia in marine sediment environments in which reactions rates might be
generally slower due to low temperatures (40).
93
Discussion
4.2.4
Activity assays and shotgun proteomics show the same reductive dehalogenases
transform several halogenated compounds
In the current study it was investigated whether the same enzymes are involved in debromination
or dechlorination of halogenated aromatic compounds and could therefore explain the broad electron
acceptor range of strain CBDB1. In addition, marine organohalide-respiring Dehalococcoidia could
occupy a broader niche in non-contaminated marine sediments if the same reductive dehalogenases are
able to transform aromatic compounds with different types of halogen substituents. Specific activities
obtained in the microtiter plate assay after cultivation of D. mccartyi strain CBDB1 with
trichlorobenzene or brominated benzenes suggested the same reductive dehalogenase enzymes are
involved in dehalogenation of brominated and chlorinated benzenes. In addition brominated phenols,
hydroxy- and methoxybenzoic acids and chlorinated thiophenes and benzonitriles were dehalogenated
by
reductive
dehalogenases
expressed
during
cultivation
with
1,2,3-trichlorobenzene
or
hexachlorobenzene. This is in good accordance with previous studies conducted with halogenated
aliphatic compounds and the purified TCE reductive dehalogenase of D. mccartyi strain 195 (95).
However, in previous transcription and expression studies with different D. mccartyi strains, a
simultaneous expression of several reductive dehalogenases when cultivated on one single electron
acceptor was observed (102, 103, 107, 108, 238). Therefore, reductive dehalogenase expression was
investigated in the current study by shotgun proteomics with strain CBDB1 cells grown with
brominate benzenes to test whether the same reductive dehalogenases previously shown to transform
chlorinated benzenes (104) can be identified after cultivation with brominated benzenes. A
simultaneous expression of four to six reductive dehalogenases was observed. The most abundant
reductive dehalogenase expressed after cultivation with bromobenzenes was CbdbA80 according to
obtained emPAI values. The emPAI value allows for an estimation of the relative abundance of a
protein (172). Indeed, CbdbA80 was previously shown to be expressed in strain CBDB1 after
cultivation with 1,2,3- and 1,2,4-trichlorobenzene and after cultivation with 2,3-dichlorophenol (104,
106). The reductive dehalogenase CbrA encoded by cbrA (previously referred to as cbdbA84) was
detected in the current study after cultivation with 1,3-dibromobenzene, 1,4-dibromobenzene or
1,2,4-tribromobenzene.
Previous
studies
using
the
purified
CbrA
enzyme
demonstrated
dehalogenation of 1,2,3-trichlorobenzene to 1,3-dichlorobenzene (104). The expression of CbrA in
cultures grown with brominated benzenes as electron acceptors suggests CbrA is involved in
dehalogenation of brominated and chlorinated benzenes. Interestingly, the cbrA gene sequence
branched with the reductive dehalogenases subseafloor cluster I (60) suggesting that reductive
dehalogenase detected in marine non-contaminated sediments may have similar functions (106) and
may transform brominated and chlorinated aromatic compounds. Reductive dehalogenase enzymes
with a diverse electron acceptor spectrum could allow organohalide-respiring Dehalococcoidia to
94
Discussion
exploit diverse natural organohalogens and broaden thereby the niche of organohalide-respiring
Dehalococcoidia in marine sediments.
4.2.5
Dehalogenation activity in marine sediments and sediment microcosms with
brominated compounds
Previous studies showed the presence of reductive dehalogenase homologous genes (rdh-genes)
(60, 61) in marine non-contaminated sediments. Furthermore, tetrachloroethene dehalogenation
activity was reported in sediment microcosms with tidal flat sediments of the North Sea containing
Chloroflexi (52) suggesting that organohalide-respiring Dehalococcoidia subpopulations could be
detected in marine sediments with the activity assay and dehalogenation rates could be determined. In
addition D. mccartyi strain MB and ‘Dehalobium chloroercia’ were isolated from estuarine sites (14,
22). However, when applying sediments or isolated cells directly retrieved from different marine
sediments to the 96-well microtiter plate-based assay or to the GC-FID-evaluated assay, no
dehalogenation activity was detected, despite the previous detection of Dehalococcoidia in the used
samples (47). This suggested that either reductive dehalogenase genes were not expressed, cells were
inactive, dead or that not all Dehalococcoidia cells detected via quantitative PCR depend on reductive
dehalogenation, as recent studies indicated (46, 48, 144). Another possibility is the expression of many
different reductive dehalogenase for different electron acceptors at low levels. Transformation rates
could be therefore below the detection limit of the activity assay.
In contrary to activity assays with crude marine sediments or cell extracts directly retrieved from
the environment, activity was detected with cells from marine sediment microcosms previously
enriched on 1,2,3-trichlorobenzene (provided by Camelia Algora, UFZ Leipzig) with the 96-well
microtiter plate assay. Activity studies revealed in vitro activity with different brominated compounds
indicating that reductive dehalogenases of marine dehalogenating microorganisms induced by 1,2,3trichlorobenzene catalyse debromination of different aromatic compounds, similar as observed for D.
mccartyi strain CBDB1. Previous studies showed transformation of 2,4,6-tribromophenol in
microcosms with pristine deep-sea sediments (60) and several studies showed dehalogenation of
brominated compounds in enrichment cultures from estuarine sediments by bacterial clades mostly
related to the delta-Proteobacteria (140, 141). However, no biochemical tests were conducted in these
studies to investigate whether corrinoid-dependent enzymes such as reductive dehalogenases were
involved in dehalogenation. In the current study inhibition studies with resting cells of marine
microcosms were conducted as previously described for Dehalococcoides spp. (105, 111). The
reduction of dehalogenating activity after exposure to propyl iodide and a slightly increased
dehalogenation activity after exposure to light suggested the involvement of a corrinoid-dependent
enzyme. Most of the characterized reductive dehalogenases have been shown to contain corrinoid as
the active cofactor (93, 94, 105, 109, 110), except for the 3-chlorobenzoate reductive dehalogenase of
Desulfomonile tiedjei containing iron in the catalytic centre (112).
95
Discussion
Reductive dehalogenation activity was also observed when samples were incubated at 4°C
suggesting that these enzymes may function also at low temperatures in deeper marine sediments. The
dehalogenation activity of resting cells exposed to oxygen did not decrease significantly in comparison
to cells incubated under anoxic conditions. In contrary to this, for D. mccartyi strain CBDB1 for
instance an increased stability of reductive dehalogenation activity was observed when stored under
anoxic conditions (170).
Marine sediment microcosms previously enriched with 1,2,3-trichlorobenzene were amended with
1,2,4-tribromobenzene to test for dehalogenation in culture and confirm results obtained by in vitro
activity
assays.
1,2,4-tribromobenzene
was
dehalogenated
to
a
further
extent
than
1,2,3-trichlorobenzene. The same was observed for D. mccartyi strain CBDB1 (7, 193) suggesting that
cobalamin-dependent reductive dehalogenases generally have a broader dehalogenation potential for
brominated electron acceptors than for chlorinated electron acceptors when comparing compounds
with similar molecular structures. Lower debromination activity was reported for D. mccartyi strain
BAV1 and 195 with brominated biphenyls than for chlorinated aliphatic compounds (143) however,
this might be related to the different size and molecular structures of the molecules underscoring the
importance of chemical and structural properties of the electron acceptor itself for reductive
dehalogenation.
Together the findings show that microorganisms performing reductive dehalogenation of
brominated aromatics occur in marine pristine sediments. The dehalogenation of various brominated
compounds in activity assays and in culture suggests that different organobromines are suitable natural
electron acceptors for organohalide-respiring microorganisms in marine non-contaminated sediment
sites. Organohalide respiration with organobromines may therefore explain the diversity and activity
of reductive dehalogenase in marine sediments detected by molecular methods in previous studies (60,
61) and by biochemical and cultivation-dependent methods in the current study in non-contaminated
sediments. In addition, the findings of the current study shows that marine sediments harbour the
capacity for transformation of halogenated aromatic compounds from natural but also anthropogenic
sources and may therefore play an important role in the natural halogen cycle.
4.3
Chemical properties of the halogenated electron acceptor influence reductive
dehalogenation
A multitude of halogenated compounds has been described to occur in the environment containing
different types of functional groups and halogens (139, 206, 235). Additionally, it was estimated that
15–20% of newly discovered marine natural products were organohalogens (235). However, it is not
clear which organohalogens could serve as electron acceptors for organohalide-respiring
Dehalococcoidia. Each Dehalococcoidia species and even each strain shows specific substrate spectra
(7-19). Therefore, the enzymes harboured by Dehalococcoidia species play a crucial role in its
96
Discussion
substrate range. However, results of the current study together with results of previous studies (119,
121, 124, 193) demonstrated that D. mccartyi strain CBDB1 transformed more different halogenated
compounds than reductive dehalogenases are encoded in the genome indicating a single reductive
dehalogenase transforms more than one halogenated compound. Additionally, the same reductive
dehalogenases were shown to be involved in the current study in the dehalogenation of brominated or
chlorinated benzenes. However, dehalogenation patterns and activities were seemingly influenced by
chemical properties of the electron acceptor itself as observed for instance for brominated and
chlorinated benzenes for which in vitro assays revealed that brominated benzenes were dehalogenated
to a further extent regardless of the electron acceptor used for the induction of reductive dehalogenase
expression in cultivation. To investigate potential reasons for this degree of unspecificity, chemical
properties of halogenated compounds tested in the current and in previous studies were evaluated in
more detail and correlated with observations made in cultivation and in biochemical experiments. The
evaluation of experimental results of the current study combined with results of previous studies
revealed that different substituents and functional groups strongly influence the regioselective
dehalogenation by Dehalococcoidia model strain CBDB1 and should be considered together with
enzymatic properties of the dehalogenating strain for the fate prediction of halogenated compounds
from natural and anthropogenic sources in anaerobic environments. The inclusion of chemical
descriptors allowed for a systematic classification of the chemical effects which govern reductive
dehalogenation in strain CBDB1. While the conclusions are based on experiments with D. mccartyi
strain CBDB1, the findings allow for some speculative extrapolation onto cobalamin-dependent
reductive dehalogenase enzymes in general – provided similar rules apply to all cobalamin-dependent
respiratory reductive dehalogenases.
4.3.1
The influence of secondary substituents onto reductive dehalogenation
Natural halogenated molecules contain a broad diversity of secondary halogen and non-halogen
substituents. Chlorinated compounds containing cyano groups have been detected in cyanobacteria,
sponges and fungi (235), chlorinated or brominated organohalogens containing hydroxy groups are
ubiquitously present and amino groups have been reported to occur naturally in form of halogenated
amino acids (235). In addition, amino, hydroxy and cyano groups are present in some of the most
commonly detected halogenated contaminants from anthropogenic sources. Dehalogenation pathways
of strain CBDB1 discussed in section 4.2.2, revealed different influence of substituents onto reductive
dehalogenation. Analyses of results obtained in the current study combined with results of previous
studies suggested an enhancing effect of secondary substituents in ortho- position to the halogen onto
reductive dehalogenation in the order -CN > -X > -OH > -NH2. For instance, whereas 1,2dichlorobenzene was not dehalogenated by strain CBDB1 (7), 2-chlorobenzonitrile was dehalogenated
to the non-halogenated benzonitrile (199) suggesting the cyano group of benzonitriles enhanced
reductive dehalogenation more than a secondary halogen substituent in ortho position.
97
Discussion
Figure 4-2: The influence of functional groups onto reductive dehalogenation when comparing halogenated
benzonitriles, anilines, benzenes and phenols. 1,2-Chlorobenzene was not transformed by strain CBDB1 (7)
while 2-chlorobenzonitrile was dehalogenated (199), 2,4-Dichloroanilines were not dehalogenated while 2,4dichlorphenol only partially (119) and 1,2,4-trichlorobenzene was dehalogenated to 1,4- and 1,3dichlorobenzene (7).
The halogen singly flanked by another halogen in 1,2,4-trichlorobenzene (7) was removed,
whereas the halogen singly flanked by the hydroxy group in 2,4-dichlorophenol was removed only
partially (119) and the singly flanked chlorine of 2,4-dichloroaniline was not removed at all. This
suggests that secondary halogen substituents enhance reductive dehalogenation more than the hydroxy
or amino groups of phenols and anilines and hydroxy substituents enhance reductive dehalogenation
more than amino substituents (119). This trend was also reflected in the partial charges of halogens
flanking the functional groups in ortho-position (Figure 4-2). Increasingly positive partial charges for
the halogen in ortho- position and the halogenated carbon were observed suggesting substituents
withdrawing electron density from the halogen and the halogenated carbon atom enhanced reductive
dehalogenation. In addition, this observation was corroborated by a correlation of the Hammett
substituent constants (239) with dehalogenation patterns observed for strain CBDB1 suggesting
substituents with an electron withdrawing effect enhance reductive dehalogenation. The Hammett
constants serve for meta- and para- secondary substituents, steric interference with ortho- secondary
substituents do not allow for a linear correlation. However, the results of this study combined with
previous studies suggest, the Hammett constituents might serve as a rule of thumb for the effect of a
substituent onto reductive dehalogenation in ortho position when no DFT modelled electron density
distribution data are available. Using the Hammett constants, also indications about the dissociation
state of the hydroxy group of halophenols during reductive dehalogenation can be gained. The
incomplete or slow dehalogenation of several dichlorophenols could be explained in part by a
dissociated hydroxy group, which according to the Hammett substituent constant has even weaker
electron withdrawing properties than the amino group of anilines and would therefore exhibit only a
98
Discussion
weak enhancing effect onto reductive dehalogenation. However, also toxic effects of phenols may
explain this observation (225).
Together, results of the current study combined with findings of previous studies indicate,
substituents with a negative inductive effect withdrawing electrons from the halogen and the
halogen-substituted carbon enhance reductive dehalogenation. The stronger the negative inductive
effect of the substituent, the more likely was the removal of halogen secondary substituents.
4.3.2
Chemical properties of heterocycles influence reductive dehalogenation
The influence of heteroatoms onto reductive dehalogenation was investigated with furoic acids,
thiophenes and pyridines. In natural systems oxygen, nitrogen and sulphur heteroatoms occur in
histidin, NADH, purine and pyrimidine bases, pyranose and furanose carbohydrates and many of the
ubiquitous enzymatic cofactors. Halogenated congeners of these molecules have been reported to
occur in the organic material of sediments (235). In addition, many halogenated pesticides used at
present are heterocyclic compounds. However, little is known about how a heterocyclic structure
influences reductive dehalogenation and thus how stable such halogenated heterocyclic compounds
are in anaerobic environments towards microbial degradation. In the current study, different
dehalogenation pathways observed for different types of heterocycles indicated heteroatoms influence
reductive dehalogenation pathways. While both tested chlorinated thiophenes were dehalogenated in
culture (234) and in activity assays, both tested chlorinated pyridines were not dehalogenated
suggesting the sulphur heteroatom in the five-membered heterocycle enhanced reductive
dehalogenation more than nitrogen in the six-membered heterocycle. Thiophenes are heteroaromatic
electron acceptors with electron-rich carbons because the free electron pair of the sulphur heteroatom
participates in the aromatic system of the five-membered ring. In pyridines, the nitrogen atom has
three hybridized electrons of which it donates one electron to the pi electron system but its free
electron pair does not participate in the six-membered aromatic system (240). Therefore, the nitrogen
heteroatom of pyridines exhibits no positive mesomeric effect and pyridines are considered as
electron-poor heterocycles with electron-deficient carbons (240). These properties were reflected in
the modelled partial charges of the two types of heteroaromatic electron acceptors (Figure 4-3). More
negative partial charges were observed for carbon atoms flanking the sulphur atom in thiophenes than
for carbon atoms flanking the nitrogen atom in pyridines. The chlorine-substituted carbon atoms of
2,3- and 2,6-dichloropyridine exhibited the most positive partial charge of all molecules tested. This
would suggest enhanced reductive dehalogenation according to previous mechanistic models of
reductive dehalogenation, considering an electron transfer onto the positively polarized
halogen-substituted carbon atom (90, 115, 241-245). However, the contrary was observed: the
electron-rich thiophenes with halogen-substituted carbons with a more negative partial charge than for
instance carbon atoms of pyridines were dehalogenated suggesting a positive mesomeric effect is
beneficial or at least not counteracting reductive dehalogenation.
99
Discussion
The oxygen heteroatom of brominated furoic acids did not inhibit dehalogenation, however it is
difficult to estimate whether the oxygen heteroatom enhanced dehalogenation, based on the data of the
current study. All tested molecules containing an oxygen heteroatom were brominated and so far, all
bromine substituents tested in the current study were dehalogenated by strain CBDB1, regardless of
the chemical structure of the molecule. Previous studies demonstrated however a positive influence of
the oxygen heteroatom of chlorinated dibenzo-p-dioxins onto dehalogenation catalysed by D. mccartyi
strain CBDB1 (121). Strain CBDB1 dehalogenated preferentially the chlorine atom doubly flanked by
chlorine and an oxygen heteroatom instead of chlorines doubly flanked by two chlorines. This may
have significant implications for the toxicity of a compound. For instance, due to the positive
influence of the oxygen heteroatom of dibenzo-p-dioxins, 1,2,3,7,8-pentachlorodibenzo-p-dioxin was
dehalogenated by strain CBDB1 to 2,3,7,8-tetrachlorodibenzo-p-dioxin which is the most toxic
halogenated dioxin known (121). However, the oxygen heteroatom in dibenzo-p-dioxin is not part of
the halogenated ring and could be therefore considered as methoxy substituent flanking the chlorine
substituent. Further research is therefore required to elucidate the influence of heteroatoms in different
ring structures. Results of the current study suggest that the influence of a heteroatom onto reductive
dehalogenation requires consideration of the molecule’s overall structure as much as type and degree
of halogenation.
4.3.3
The role of the halogen type in reductive dehalogenation
Natural halogenated compounds include chlorine-, bromine-, iodine-, and fluorine-containing
organic compounds (235) and iodated, brominated, chlorinated and fluorinated compounds are
introduced into the environment in form of contrasting agents, flame retardants, pesticides and
antibiotics into the environment. Results of this study demonstrate not all halogen substituents are
equally well suitable for transformation via reductive dehalogenation. When comparing brominated,
Figure 4-3: Dehalogenation reactions and partial charges of halogenated aromtics. Chlorine partial charges are
indicated in blue, fluorine partial charges in green, bromine partial charges in red and partial charges of the
halogen-substituted carbons in black.
100
Discussion
chlorinated and fluorinated molecules tested in this study, and specifically molecules substituted with
two different halogen types, such as 2-chloro-6-fluorobenzonitrile and 3-bromo-2-chloropyridine, an
enhanced removal of bromine compared to chlorine was observed, which was also reflected in the
NBO partial charges (Figure 4-3). Fluorine was not removed even though it was flanked in 2-chloro-6fluorobenzonitrile by the functional group with the strongest electron withdrawing properties of all
substituents tested so far with strain CBDB1. The enhanced removal of bromine substituents compared
to chlorine substituents supports the observation made with non-halogen substituents and heteroatoms
that the most relevant parameter for reductive dehalogenation is the withdrawal of electron density
from the halogen and not from the carbon. Otherwise, fluorine should allow for the most extensive
dehalogenation, while bromine should be removed to a lesser extent than chlorine.
This can be explained by the chemical properties of different halogen substituents. Halogens
exhibit a negative inductive effect (-I-effect) by withdrawing electron density from neighbouring
atoms with lower electronegativity and a positive mesomeric effect (+M-effect) by donating a free
electron pair participating in the aromatic system in the order I < Br < Cl < F (i.e. bromine has a
weaker negative inductive effect than chlorine but a stronger positive mesomeric effect) (246, 247).
This means that fluorine will induce a more positive partial charge on the halogenated carbon than for
instance bromine. Results of the current study demonstrated the less electronegative a halogen was, the
more likely was its dehalogenation by strain CBDB1. A weaker aryl-bromine bond compared to the
aryl-chlorine bond (236, 237) could additionally facilitate reductive dehalogenation.
For some molecules tested in the current study, abiotic dehalogenation of bromine was observed
and lower growth yield of strain CBDB1 were obtained suggesting an optimum range for the strength
of the aryl-halogen bond is required to allow for energy generation via reductive dehalogenation. This
could be especially true for iodinated compounds with an even weaker carbon-iodine bond compared
to the carbon-bromine bond. Therefore, abiotic dehalogenation under reduced conditions such as
observed for brominated aromatic compounds could compete with biotic dehalogenation reactions by
organohalide-respiring Dehalococcoidia species.
4.3.4
Chemical properties governing dehalogenation pathways of strain CBDB1 suggest
halogen–cobalamin interaction during reductive dehalogenation
Previous studies suggested that reductive dehalogenation reactions are nucleophilic substitutions
in which the C-X bond cleavage is initiated by a B12CoI attack at the carbon site and in which an
intermediate Co–C bond is formed (90, 115, 241-245). The findings of this study however indicate
that the site of interaction is rather the halogen than the carbon for the following reasons:
The aromatic pyridine heterocycle is considered as an electron-poor ring-system suggesting
enhanced transfer of electrons on carbon flanking nitrogen. Thus a nucleophilic attack of cobalamin on
the halogen-substituted carbon for 2,3-dichloropyridine or 2,6-dichloropyridine should be facilitated.
However, the contrary was observed. The electron-poor pyridine was not dehalogenated while the
101
Discussion
electron-rich heterocycle thiophene was dehalogenated. Furthermore, if halogens were removed by a
nucleophilic attack onto the substituted carbon atom the reactivity of halogens for a nucleophilic attack
(SNAr) should increase with increased CAr–X bond dissociation energy in the order F > Cl > Br > I
(248), due to an increasing electron deficiency at the aromatic halogen-substituted carbon as site of
nucleophilic attack. However the opposite reactivity in the order Br > Cl > F during reductive
dehalogenation was observed in the current study. Instead, the reactivity order correlated with
reduction potentials reported for monohalogenated benzenes yielding the order Br > Cl > F with Br
having the least negative reduction potential (249). In addition, the regioselective dehalogenation
catalysed by strain CBDB1 was predicted by the most positive partial charge of the halogen and not by
the most positive partial charge of a halogen-substituted carbon.
Therefore, instead of an electron transfer to the halogen-substituted carbon in a nucleophilic
substitution reaction, a single electron transfer (250) onto the halogen may be considered. CoI and
other transition metals with a singlet d8 configuration were shown to act as Lewis bases, and thus can
form hydrogen bonds with an electron accepting compound (251-254). A halogen could function as
such an electron accepting Lewis acid, due to a region with positive electrostatic potential. This region
with positive electrostatic potential is called -hole (255). It could interact with the lone electron pair
of a Lewis base (256-258). Therefore, the halogen and the CoI could potentially interact as Lewis base
and Lewis acid, respectively (personal communication, Prof. Dr. Schüürmann, Department for
Ecological Chemistry, UFZ, Leipzig).
The -hole is progressively larger and more positive for iodine > bromine > chlorine.
Additionally the presence of the -hole depends on the environment of the halogen atom in the
molecule and requires electron density withdrawing atoms in the molecule (255). The improved
prediction rates of dehalogenation pathways catalysed by strain CBDB1 using sigma partial charges
compared to pi partial charges indirectly support the hypothesis of the involvement of the apical
-hole of halogen substituents of aryl compounds. Interestingly the -hole does not occur in fluorine
(255) suggesting that the absence of this region prevents reductive dehalogenation of fluorinated
molecules. However, this will require further studies with diverse fluorinated aromatic compounds.
It remains to be elucidated why electron-poor pyridines are not reductively dehalogenated.
Electron-poor pyridines should be more reactive to the formerly proposed SNAr reaction mechanism
due to a more positively polarized halogen-substituted carbon but also to the newly proposed single
electron transfer because the molecule exhibits an overall larger electron affinity. Nevertheless, the
reduced reactivity of pyridines towards reductive dehalogenation emphasise the importance of the
carbon–halogen bond polarization for reductive dehalogenation.
The direct interaction of the cobalamin cofactor with the halogen is supported by recent
descriptions of the crystal structure of two reductive dehalogenases. Electron paramagnetic resonance
measurements and modelling with a reductive dehalogenase from the aerobic strain Nitratireductor
102
Discussion
pacificus strain pht-3B indicated a direct cobalamin-halogen interaction (117). Additionally, for the
reductive dehalogenase of Sulfurospirillum multivorans a very small pocket containing the active site
was reported (259), which suggests that an electron transfer onto the halogen would be facilitated
compared to the transfer onto the shielded carbon atom. For the electron acceptor range of
Dehalococcoidia in marine sediments a cobalaminhalogen interaction could allow for the usage of a
broad range of different halogenated molecules with different structures. However, a cobalamin–
halogen interaction implies that organohalide-respiring Dehalococcoidia cannot evade organohalogens
for respiration with the encoded set of reductive dehalogenases and suggests that organohaliderespiring Dehalococcoidia cannot substitute halogenated compounds by compounds with halogen-like
functional groups such as for instance pseudohalogens. Previous studies conducted in this research
group corroborate this hypothesis by showing that several tested non-halogenated compounds did not
allow for growth of strain CBDB1 (260).
4.3.5
Implication for other reductively dehalogenating microorganisms
While findings of the current study suggest a halogen–cobalamin interaction for reductive
dehalogenation catalysed by strain CBDB1, it remains to be elucidated to which extent these rules
apply to different dehalogenating strains. Among the D. mccartyi strains different dehalogenation
patterns can be observed, and even more distinct patterns have been reported for different
Dehalococcoidia species and other reductively dehalogenating microorganisms. The prediction of
dehalogenation pathways by using the most positive halogen partial charge served for strain CBDB1
but will need to be evaluated for different dehalogenating strains with different dehalogenation
patterns. Other strains may require different chemical parameters for a successful description and
prediction of their dehalogenation patterns and a correlation of the chemical descriptors with substrate
spectra of purified reductive dehalogenases will be required.
However, rules identified for strain CBDB1 may allow a deeper understanding of reactivity
patters of dehalogenating strains despite of variations observed for dehalogenation patterns of different
bacterial strains. D. mccartyi strain DCMB5 for instance shows similar dehalogenation patterns as
strain CBDB1 for chlorinated benzenes and dibenzo-p-dioxins (261). Like strain CBDB1, strain
DCMB5 preferentially dehalogenated the chlorine atom of 1,2,4-trichloro-dibenzo-p-dioxin flanked by
two electron withdrawing groups suggesting rules identified for strain CBDB1 account also for other
D. mccartyi strains performing hydrogenolytic dehalogenation. Additionally, strain DCMB5 did not
dehalogenate
the
isolated
chlorine
substituent
of
2-monochlorodibenzo-p-dioxin
or
1,4-dichlorobenzene but the chlorine substituent of 1-monochlorodibenzo-p-dioxin yielding a nonhalogenated reaction product (261), demonstrating the importance of electron density withdrawal from
the halogen by flanking substituents or heteroatoms for reductive dehalogenation. For D. mccartyi
strain 195,
a
dehalogenation
of
singly
flanked
chlorine
substituents
was
reported
for
1,2,3,4-tetrachlorobenzene or 1,2,3-trichlorobenzene. However, also strain 195 seems to require
103
Discussion
functional groups withdrawing electron density from the halogen. For instance, strain 195
dehalogenated 2,3,4,6-tetrachlorobiphenyl to 2,4,6-trichlorobiphenyl, preferentially removing doubly
flanked chlorine substituents similar to strain CBDB1, while 2,4,6-trichlorobiphenyl was not
dehalogenated by strain 195 (205). Also, strain 195 was not able to dehalogenate isolated chlorine
substituents. Therefore, dehalogenation patterns of different D. mccartyi strains suggest, that generally
functional groups withdrawing electron density from the halogen enhance reductive dehalogenation,
although the presence of different reductive dehalogenases in the genome of DCMB5 and 195 may
lead to different dehalogenation patterns. It remains therefore to be elucidated to which extent the
enzymatic properties of reductive dehalogenases compared to the chemical properties of the electron
acceptor influence the dehalogenation pattern of organohalide-respiring microorganisms.
4.3.6
Potential applications and usage of the findings for fate prediction of halogenated
compounds in the environment
The importance of substituent influence on the potential for reductive dehalogenation has
practical impact. Continuously new halogenated compounds are developed for which an experimentalbased assessment of degradation products is time and cost intensive. The analysis of chemical
parameters that influence reductive dehalogenation could help to better understand and predict the fate
of halogenated compounds in anoxic environments and improve remediation strategies building upon
organohalide-respiring microorganisms.
Halogenated aromatic compounds such as chlorpyrifos methyl, bromacil, chlorothalonil,
triclocarban or triclosan, atrazin, bromoxynil, dichlobenil, and polybrominated diphenylethers contain
diverse non-halogen functional groups and/or heteroatoms and are currently used as pesticides, wood
protectants, flame retardants, disinfectants or pharmaceuticals. The functional groups investigated in
the current study are present in many of these halogenated compounds and a better understanding of
their transformation and degradation pathways in anaerobic environments will allow for improved fate
prediction. For instance, imidacloprid is one of the most commonly used neonicotinoid pesticides used
at present (262) and is suspected to be involved in the currently observed bee dying (263). The six
membered heterocycle of imidacloprid contains a nitrogen heteroatom and a chlorine substituent
similar to the chlorinated pyridines tested in the current study. According to the findings of the current
study using chlorinated pyridines, imidacloprid is likely not dehalogenated by D. mccartyi strain
CBDB1. If these findings are extrapolated onto other dehalogenating strains it can be speculated that
such compounds may accumulate in anaerobic environments such as for instance aquifers. The
neonicotinoids clothianidin and thiamethoxam however, are chlorinated five-membered sulphur- and
nitrogen-containing heterocycles. According to the results of the current study a dehalogenation by
D. mccartyi stains can be predicted. However, further research is required to establish an
experimentally acquired basis for such prediction systems. If the approach of this study will be
extended onto more dehalogenating strains and diverse halogenated compounds, recommendations for
104
Discussion
the usage or treatment of specific halogenated chemicals could be made. In addition, potential toxic
effects of functional groups, as observed in the current study for compounds containing hydroxy
substituents, will need to be included into such prediction systems to take into account adverse side
effects of the halogenated chemical itself onto the dehalogenating bacterial community. For instance,
the pharmaceutical oxyclozanid or the disinfectant triclosan could exhibit growth-inhibiting effects
onto dehalogenating microorganisms such as strain CBDB1 due to the hydroxy substituents. This
could diminish the success of bioremediation strategies using reductively dehalogenating bacteria.
In addition, some D. mccartyi strains were described to produce the very toxic vinyl chloride
during reductive dehalogenation (8, 11). These examples demonstrate that remediation strategies
building upon reductive dehalogenation by D. mccartyi strains need careful evaluation according to
the contaminants present in the soil and the expected reaction products. For this, a prediction system
based on chemical descriptors correlated with dehalogenation patterns of particular dehalogenating
strains can be highly valuable for a first in silica assessment of potential reaction products. In addition,
such prediction systems may allow improved fate prediction of halogenated contaminants by naturally
occurring dehalogenating microorganisms when combined with bio molecular or mass spectrometry
tools, allowing for the identification of strains and reductive dehalogenase genes or even proteins in
contaminated sites.
4.4
Non-halogenated electron acceptors of Dehalococcoidia in marine sediments
Genome annotation of two Dehalococcoidia single-cells (46, 48) and Dehalococcoidia
metagenomes
(144)
revealed
metabolic
features,
which
allow
for
an
organohalide
respiration-independent mode of living. In Dehalococcoidia SAG-C11 from sediments of the Aarhus
Bay a gene coding for a reductive dehalogenase subunit A was identified however a TAT leader
sequence and a gene coding for the membrane anchor RdhB were not detected suggesting the RdhA is
not involved in respiratory functions in SAG-C11. However, genes coding for dissimilatory sulphite
reductase subunits were identified (personal communication, Dr. Kenneth Wasmund, UFZ, Leipzig).
The reductive type of dissimilatory sulphite reductases have been described in the bacterial phyla
Proteobacteria (Delta-Proteobacteria), Firmicutes, Nitrospirae, Thermodesulfobacteria, Caldiserica
and Actinobacteria, and the archaeal phyla Euryarchaeota and Crenarchaeota and the candidate
division Aigarchaeota (146-149, 202) but never in the phylum Chloroflexi. In addition, phylogenetic
analysis of environmental dsrAB sequences revealed many lineages without any cultivated
representatives suggesting that several dissimilatory sulphite reducing microorganisms have not yet
been identified (150, 201). Furthermore, sediment sites in which high sulphate reduction activity was
described did not reveal high dsrA gene copy numbers or 16S rRNA genes of sulphate reducing
prokaryotes (49, 163) suggesting that currently available primers do not amplify full dsr gene
diversity. Primers used in the current study allowed for the amplification of dsrA, dsrAB or dsrN
105
Discussion
sequences which clustered together with the dissimilatory sulphite reductase genes of
Dehalococcoidia SAG-C11 (Figure 3-18 and Figure 3-19) indicating the phylum Chloroflexi
contributes to dsr gene diversity and suggesting that dissimilatory sulphite reduction occurs in a
broader variety of prokaryotes than previously recognized.
The occurrence and diversity of dissimilatory sulphite reductase genes related to Chloroflexi was
investigated by molecular tools because previous attempts to enrich Chloroflexi by cultivation in the
current and in previous studies remained unsuccessful. For the molecular investigation different
sediments were chosen in which the Dehalococcoidia subgroup of SAG-C11 was detected. SAG-C11
belongs to the DSC-GIF3-B subgroup and was shown to represent about 4% of Dehalococcoidia 16S
rRNA gene sequences in the sediment core from 10 to 300 cmbsf at the Aarhus Bay site (47). The
DSC-GIF3-B subgroup was also described in the sediment core 371 of the Baffin Bay (47). It was
suggested that similar biogeochemical conditions at the two sites might explain the similar distribution
(47). Sediments from the core of the Bay of Aarhus and core 371 from the Baffin Bay exhibited
similar TOC ranges through the depths of the cores (47, 200). Most of the analysed DsrA or DsrB
sequences obtained in the current study from sediments of the Aarhus Bay, the Baffin Bay and the
DsrA sequence obtained from the Wadden Sea, formed stable cluster with DsrA or DsrB sequences of
Dehalococcoidia SAG-C11. The occurrence of the DSC-GIF3-B subgroup in different sediment sites
(47) suggests the potential for sulphite reduction by members of the class Dehalococcoidia could be
even more widespread in different sediment sites and could be investigated by primers developed in
the current study.
Several dsr sequences amplified in the current study derived from sediments of the Bay of Aarhus
affiliated with environmental sequences previously termed ‘uncultured DrsAB lineage 3’ (150). In
addition, two of the amplified sequences from the Baffin Bay affiliated with DsrA or DsrB sequences
previously termed ‘uncultured DsrAB lineage 2’. ‘Uncultured dsrAB lineage 2’ contains dsrAB
sequences derived from sediments of 500 cmbsf with very low sulphate concentrations (152). For
these sequences, it remains to be elucidated whether they belong to the phylum Chloroflexi or to a so
far unidentified bacterial division.
Beside phylogenetic analysis of the dsr genes by different treeing methods, additional evidence
for linking dissimilatory sulphite reductase genes amplified from marine sediments with
Dehalococcoidia was gained by the analysis of luxR genes co-amplified with dsrAB. In SAG-C11 dsr
genes were flanked on both sides by genes previously described in Dehalogenimonas (Figure 3-16
page 69), including the luxR gene and genes involved in siroheme synthesis, required for bacterial
sulphur metabolism (159). All obtained LuxR amino acid sequences amplified on one fragment with
dsrAB had highest sequence similarity to LuxR of SAG-C11 suggesting dsrAB sequences amplified
with luxR belong to Dehalococcoidia.
Additionally, a fragment coding for dsrABDN was amplified. Because this fragment did not code
for any marker genes previously identified in Dehalogenimonas an additional link between
106
Discussion
dissimilatory sulphite reductase genes amplified from sediments and dissimilatory sulphite reductase
genes detected in Dehalococcoidia SAG-C11 was provided by analysis of the dissimilatory sulphite
reductase gene order within the amplified fragment. Different organization of the dissimilatory
sulphite reductase genes within different sulphate reducing Bacteria or Archaea have been described
previously (156). For instance, in Thermodesulforhabdus norvegica dissimilatory sulphite reductase
genes follow the order dsrD-dsrA-dsrB-dsrN-dsrC (264), whereas in Desulfotomaculum acetoxidans
the order is dsrC-dsrN-dsrA-dsrB-dsrD, in Syntrophobacter fumaroxidans dsrA-dsrB-dsrN-dsrC. In
Archaeoglobus fulgidus a so far uncharacterized archaeal protein succeeds dsrA and dsrB and drsD.
Sequencing of several fragments amplified from marine sediments with internal primers in the current
study revealed the same organization of dsr genes as detected in DEH-SAG-C11, however dsr gene
organization was also similar to the dsr gene organization in Caldiserica. Both show a dsr gene order
of dsrA-dsrB-dsrD-dsrN-dsrC-dsrM-dsrK (202) of which dsrA to dsrC were amplified within the 3.9
kb fragment (Figure 3-16) from marine sediments in the current study. The genes coding for DsrJOP,
detected in all genomes of sulphate reducing organisms so far analysed were not identified in the
genome of SAG-C11 and the Gram-positive bacteria, Caldivirga maquilingensis (156, 265) and
Caldiserica (202). Moreover, DsrA, DsrB and DsrN sequences of Caldiserica sp. affiliated closely in
phylogenetic analysis with the DsrA, DsrB or DsrN sequences amplified from sediments in the current
study. Direct comparison of dsr genes sequences amplified from sediments with dsr sequences of
Caldiserica and DEH-SAG-C11 revealed higher sequence identities for dsr genes of SAG-C11 than
for dsr genes of Caldiserica, suggesting dissimilatory sulphite reductase genes amplified from
sediments origin from Dehalococcoidia. In addition, the GC content of dsrAB fragments amplified in
the current study is highly similar to the one of the SAG-C11 genome whereas the GC content is more
than 10% different to the GC content of the genome of Caldiserica. Interestingly, dsrAB genes of
Caldiserica showed also high GC content divergence to their own genome with a GC content of the
genome of 35% compared to the GC content of Caldiserica dsrAB of 44.1%. Caldiserica sp. may have
acquired dsr genes via a horizontal gene transfer (150). Possibly, Caldiserica sp. could have acquired
its dsrAB sequence from a Chloroflexi-related ancestor or both species acquired dsr genes by
horizontal gene transfer from a Firmicutes related ancestor.
The comparison of dsrAB sequences amplified in the current study with dsrAB sequences of
SAG-C11 suggested, at least another species and possibly another genus level within the class
Dehalococcoidia harbours the potential for dissimilatory sulphite reduction. This was deduced from
correlation of dsrAB sequence identities with 16S rRNA identities as described previously (150, 203).
16S rRNA and DsrAB phylogenies have been described as largely congruent, except for several
distinct lateral gene transfers (150) and evolutionary relationships may be inferred to a certain extent
based on the dsrAB gene sequences, similar to the ones inferred from 16S rRNA sequences (266, 267).
Therefore, dsrAB sequences have been used as phylogenetic marker to investigate sulphate reducer
diversity (151, 157, 158, 268-270). A 16S rRNA gene sequence similarity of less than 99% was
107
Discussion
proposed as threshold for delineating species level operational taxonomic units (266). Based on this a
dsrAB sequence identity of less than 90% was recommended as a threshold which will likely represent
two different species (150). Analysed dsrAB sequences amplified in the current study revealed 70% to
100% sequence identity with dsrAB of SAG-C11 suggesting dsr genes of another Dehalococcoidia
species were amplified in the current study. In addition, 71% to 87% dsrAB gene identity has been
related to 86.5% to 94.5 % 16S rRNA identity (150). Yarza and colleagues suggested that a 16S rRNA
sequence identity of 94.5% or lower provide strong evidence for two distinct genera (267). Therefore
it seems likely that dsr sequences of another Dehalococcoidia genus were amplified in the current
study.
Together the amplification of diverse dissimilatory sulphite reductase genes related with
Chloroflexi indicated that the potential for sulphite respiration is not restricted to one species within
the class Dehalococcoidia, phylum Chloroflexi. The findings provide further evidence that the class
Dehalococcoidia is metabolically versatile and contribute to a deeper understanding of the ubiquitous
distribution of Dehalococcoidia in marine sediments and the stratification of Dehalococcoidia
according to bio geo chemical gradients in diverse sediment environments (47).
4.5
Implication for Dehalococcoidia from marine sediments
The broad range of halogenated electron acceptors of strain CBDB1 identified in the current and
in previous studies (7, 118, 119, 121, 124, 193) exceed the number of reductive dehalogenases present
in the genome of strain CBDB1 (26, 29), indicating that each reductive dehalogenase catalyses the
dehalogenation of various halogenated molecules. The interaction of the cobalamin cofactor with the
halogen substituent instead of the halogen-substituted carbon might allow for a less specific
interaction of the reductive dehalogenase with the substrate. A broad spectrum of halogenated
compounds as electron acceptors for organohalide-respiring Dehalococcoidia suggests that a multitude
of natural organohalogens in the environment could serve as natural halogenated electron acceptor for
respiration and growth of Dehalococcoidia even if the individual halogenated compounds are present
in very low quantities. Haloorganic soil humic acids (271, 272) were detected for instance in humic
forest lake sediments (273) and peatlands (274). In addition, a huge diversity of brominated and
chlorinated compounds produced by marine and terrestrial organisms (235) and even bacteria (275)
were described to contain similar or the same chemical structures as electron acceptors tested in the
current study. Halogenated electron acceptors introduced into sediments by precipitation and decay of
dead marine organisms or by living organisms releasing brominated compounds into the sediments
(227) could allow for organohalide respiration at low rates and continuous metabolic activity of
Dehalococcoidia. Together, these compounds could provide an ecological niche for Dehalococcoidia
subgroups depending on organohalide respiration. Members of the subgroup “Ord-DEH” to which all
so far isolated D. mccartyi strains belong was shown to occur in marine sediments mostly in upper
108
Discussion
sedimentary levels, except for sediments from the Peru margin where this group was detected up to 30
mbsf (47). Site-specific variation might be also due to different sediment types, which accumulated
over geological time scales. An investigation of the organohalogen content of these sites could
elucidate this question. However, the detected reductive dehalogenase diversity in marine sediments
(60, 61) does not necessarily indicate diversity of Dehalococcoidia species as even one single strain
can harbour many reductive dehalogenase genes (26, 29). Therefore, the investigation of rdhA genes
and would need to be combined with analysis of 16S rRNA genes or single cell approaches to learn
more about the diversity of organohalide-respiring Dehalococcoidia in marine sediments.
The analysis of metabolic features of Dehalococcoidia derived from marine sediments in previous
studies revealed Dehalococcoidia are metabolically versatile (46, 48, 144) and the findings of the
current study demonstrated that the potential for organohalide-independent respiration is more
widespread in the class Dehalococcoidia. Possibly, the enrichment of Dehalococcoidia with an
organohalide respiration-independent mode of living remained so far unsuccessful because strongly
selecting cultivation conditions have not been identified so far. The organohalide-respiring mode of
living of cultivated members of the class Dehalococcoidia provided the possibility to use
organohalogens as a selecting parameter for enrichment and isolation. Because isolated
Dehalococcoidia strains grow slowly and to only low cell densities, an enrichment of their marine
relatives with strategies using more common and less toxic electron acceptors may be extremely
difficult. Faster growing bacterial species might outcompete Dehalococcoidia. Possibly cultivation of
single cells could provide a tool to cultivate Dehalococcoidia with an organohalide respirationindependent mode of living (276, 277).
109
Conclusion
5
Conclusion
Dehalococcoidia have been shown in the current study to dehalogenate diverse halogenated
electron acceptors containing different halogen and non-halogen substituents and heteroatoms. The
number of identified electron acceptors exceeds the number of reductive dehalogenase homologues in
the genome of strain CBDB1 indicating single reductive dehalogenases transform several halogenated
substrates each. A low substrate specificity of reductive dehalogenases leading to a versatile electron
acceptor range could be explained in part by the cobalamin–halogen interaction proposed in the
current study. The diverse range of halogenated electron acceptors transformed by Dehalococcoidia –
shown in the current study for D. mccartyi strain CBDB1 – would allow organohalide-respiring
Dehalococcoidia to occupy a broader niche in natural environments than anticipated. Growth with
brominated electron acceptors shown for the model strain CBDB1 in the current study suggests
brominated compounds could serve in marine sediments to sustain growth of Dehalococcoidia
depending on organohalide respiration. For Dehalococcoidia growing independently from
organohalide respiration, sulphite respiration could contribute to the broad diversity and stratification
of Dehalococcoidia along geochemical gradients. Functional genes associated with sulphite
respiration were linked to Dehalococcoidia in the current study and were shown to be diverse and
distributed in various sediment environments. Together the diverse electron acceptor range and modes
of respiration catalysed by Dehalococcoidia described in the current study allow for a better
understanding of the ubiquitous and abundant occurrence of Dehalococcoidia in marine sediments. In
addition, the findings of the current study may allow for enhanced fate prediction of halogenated
compounds from natural and anthropogenic sources in anaerobic sediment environments.
110
Outlook
6
Outlook
To further explore growth of Dehalococcoidia with natural halogenated compounds, the
cultivation of strain CBDB1 may be expanded in the future onto halogenated molecules with more
complex ring structures. In addition, the expression of enzymes during cultivation with complex
structures and with compounds containing different functional groups may reveal further reasons for
the reductive dehalogenase diversity harboured in genomes of D. mccartyi strains. The role of the
reductive dehalogenase diversity will be also clarified by recent successful heterologous expressions
of functional reductive dehalogenases (99), which will allow for a further electron acceptor
characterization of single purified reductive dehalogenases. These findings can be combined with the
chemical descriptors identified in the current study to verify and compare the influence of enzymatic
vs. chemical properties of the electron acceptor. A special focus could be placed onto different
heteroaromatics with the same heteroatom integrated in different ring systems, such as for instance
electron-poor pyridine and electron-rich pyrrol, to elucidate further the mechanism governing
reductive dehalogenation. The halogenation of large and complex ring structures such as humic acids
by haloperoxidases and subsequent cultivation of strain CBDB1 may provide further information
about the significance of a halogencobalamin interaction for the usage of large and complex aromatic
organohalogen structures.
Additionally, the influence of functional groups onto dehalogenation pathways but also growth
should be studied regarding toxic and inhibitory effects as observed in the current and in previous
studies for the hydroxy group. Other functional groups may exhibit toxic effects similar to the hydroxy
group of phenols. To investigate toxic or inhibitory effects further, D. mccartyi strain CBDB1 could be
monitored for changes on the mRNA or protein expression level using for instance halogenated and
non-halogenated anisols and phenols in parallel cultivation experiments. In addition, an evaluation of
toxic effects of obtained dehalogenation products onto the dehalogenating strain could be investigated
by addition of these reaction products to cultures together with a non-toxic halogenated electron
acceptor and comparison of growth and dehalogenation rates. Furthermore, ecotoxicological assays
with the transformation products of reductive dehalogenation by Dehalococcoides strains using
prokaryotic and eukaryotic model systems could allow for a better understanding of the benefit of
dehalogenation reactions for a reduction of toxicity of halogenated contaminants.
Also the combination of various strains with specific functions permitting the mineralization of
the tested halogenated compounds could be an interesting field of investigation.
For the activity assay a more extensive investigation of the influence of the electron acceptor used
during cultivation onto activity measured in the in vitro assay and a standardization of the cell growth
status at the moment of cell harvest could help to standardize the activity assay further. At present
measured activities are related to whole cell proteins, however this does not necessarily reflect the
amount of reductive dehalogenases expressed at the moment of cell harvest and the activity of the
111
Outlook
enzymes according to the growth phase of the microbial inoculum. This introduces variations in
measured activities, even though not relative but absolut. However, the usage of single purified
reductive dehalogenases will permit a quick improvement and further standardization of the activity
assay.
Furthermore, the partial charge model developed in the current study will need to be verified for
its stability towards changes of the basis sets and for an even broader electron acceptor range. Here a
special focus should be placed on the identification of a threshold predicting whether a compound is
dehalogenated or not by a specific strain. Possibly, such identification is only possible for one
substance class and not for many different. Therefore, the investigation of dehalogenation pathways of
different highly halogenated compounds could provide further experimental data as a basis for the
evaluation of chemical descriptors. When purified reductive dehalogenases are available also a
comparison of dehalogenation rates in activity assays with partial charges will lead to a better
understanding of the importance of partial charge distributions onto dehalogenation. A broad set of
different dehalogenating strains of the species D. mccartyi, other dehalogenating members of the class
Dehalococcoidia and different other dehalogenating microorganism will require further studies using
different chemical descriptors to identify which parameters influence reductive dehalogenation for
other strains with different dehalogenation patterns. This may lead to different mechanistic models in
other microorganisms.
For Dehalococcoidia from marine sediments the newly gained information about their versatile
metabolism offers the possibility to set up long-term cultivation experiments designed for specific
Dehalococcoidia subgroups. For Dehalococcoidia subgroups that are not depending on organohalide
respiration, cultivation set-ups using the genomic information from single amplified genomes of
Dehalococcoidia could be conducted and dissimilatory sulphite reductase activity could be tested by
using sulphate, sulphite or organosulphonates as electron acceptors. For organohalide-respiring
Dehalococcoidia brominated compounds could be used for enrichment, as they may allow
organohalide-respiring Dehalococcoidia to grow even if reductive dehalogenases with less reduction
potential than those of strain CBDB1 are encoded in their genomes.
An additional interesting field could be the investigation of the role of cytoplasmic reductive
dehalogenase subunit A, which has been detected in SAG-C11 but also in Dehalogenimonas strains
(20). Both strains possess LuxR transcriptional regulator, which have been suggested to be inhibited
for instance by halogenated furanones produced by marine algae (132). The half-life of the protein
LuxR was shown to be reduced 100-fold by halogenated furanones (132). An investigation of
cytoplasmic reductive dehalogenase homologs for their potential role in detoxification of molecules,
or in providing aromatic compounds for degradation pathways could elucidate the role of reductive
dehalogenase likely active in the cytoplasm of members of the Dehalococcoidia.
112
Supplement
Table 7-19: Accession numbers of dissimilatory sulphite reductase sequences obtained in this study
Clone
Accession number
Aarhus_Bay_dsr_clone_AA001_dsrA
KU561069
Aarhus_Bay_dsr_clone_AA142_dsrA
KU561070
Aarhus_Bay_dsr_clone_AA209_dsrA
KU561071
Aarhus_Bay_dsr_clone_AA215_dsrA
KU561072
Aarhus_Bay_dsr_clone_AA285_dsrA
KU561073
Baffin_Bay_dsr_clone_BB022_dsrA
KU561074
Baffin_Bay_dsr_clone_BB048_dsrA
KU561075
Wadden_Sea_dsr_clone_WS013_dsrA
KU561076
Aarhus_Bay_dsr_clone_AA240_dsrA
KU561077
Aarhus_Bay_dsr_clone_AA252_dsrA
KU561078
Aarhus_Bay_dsr_clone_AA271_dsrA
KU561079
Aarhus_Bay_dsr_clone_AA14E_dsrA
KU561080
Aarhus_Bay_dsr_clone_AA28G_dsrA
KU561081
Aarhus_Bay_dsr_clone_AA20H_dsrA
KU561082
Aarhus_Bay_dsr_clone_AA020_dsrA
KU561083
Aarhus_Bay_dsr_clone_AA075_dsrA
KU561084
Baffin_Bay_dsr_clone_BB014_dsrA
KU561085
Aarhus_Bay_dsr_clone_AA215_dsrB
KU561086
Aarhus_Bay_dsr_clone_AA285_dsrB
KU561087
Baffin_Bay_dsr_clone_BB022_dsrB
KU561088
Aarhus_Bay_dsr_clone_AA026_dsrB
KU561089
Aarhus_Bay_dsr_clone_AA252_dsrB
KU561090
Aarhus_Bay_dsr_clone_AA271_dsrB
KU561091
Aarhus_Bay_dsr_clone_AA20H_dsrB
KU561092
131
Supplement
Sequences obtained with internal primers
>Aarhus_clone_290
ATCATGGTGGTATAGTTGGCGTTAGGGGCTATGGGGGAGGGGTTATAGGTAGGTACTCCGATGTCCCCGAGCAATTCCCA
AATGTAACTGCCTTCCACACTATAAGGGTAAATATGCCCAGTGGCTGGTTCTACACCACTAAGGCTCTTAGAGGTGTCTG
TGATGTCTGGGAAAAATATGGTAGCGGTCTCACTAATCTCCACGGGGCGACAGGTGACATCATACTTTTGGGAACGACTT
CGGAAAATTTACAGCCTTGTTTTGACGCACTGAGTGATGAGGCGGGATTTGATTTGGGGGGTTCTGGCTCAGTCTTGAGG
ACGCCAAGCTGTTGTGTGGGACCGGCTAGATGTGAATGGTCCTGCATAGACACTCTTGACATATGTAACGACCTGACTCA
TGAATTTCAGGACGAGCTACACCGACCCATGTGGCCATATAAATTCAAAATTAAGATATCCGGATGCCCCAATGACTGTG
TTGCCGCCATTGCTAGGGCTGATATGCCCATAATCGGCACCTGGCGTGATTACCTTAGAGTAGACCAGGATGAAGTGAGA
AAATATGTAGCTAATGGCTTCGATATCCAGAGAGAAGTTATCGCTATGTGTCCCACCTGGGCACTGGACTGGGATGAAAA
AGCACAGGAGCTCAAGGTGAAGCAAGAAGAATGTGTCAGGTGCATGCATTGTATTAACCGAATGCCTAAGGCAATCAGGC
CAGGGGTTGAGCGAGGAGCTACCATTCTTATCGGTGGTAAAGCGCCGATAATAAAGGGGGCACTGCTTTCCTGGGTGCTG
GTTCCCTTTATGAAGATGGAGCCACCCTATACCGAATTCAAGGAGCTGGCACGCAAAATATGGGAATGGTGGGACGAAAA
TGGCAGGACAAGGGAAAGGGTTGGCGAACTCATTGAGAGATTGGGCATGGCGCAGTTCCTGCGAGAAATGGGACTAAAGC
CAATCCCTCAAATGGTGTTCCGACCCAGGTCCAACCCGTATGTCTTCTGGCCCCCAGAAAAGAGGAGAAAGTAAAGGGAA
TAAAATAAATCAGTTGAGGAGAGCGCTTTGACTATTGATATTGGTCCTCCAGATTATAAGCTTATGCTTCACCCAGTAAT
CAAGGAAAACTATGGCAAGTGGAAATACCATGAGATATTAGAGCCTGGTGTGCTGAAGCACGTATCTGAGACCGGGGGCG
AGGTATATACAGTACGAGTTGGCTCGCCCAGGCTGGTAAGCATTGACTTCATACGGGAGATTTGTGATATCGCTGACAAA
TACTGTGATGGTTACTTAAGGTTTACCAGTCGGCATTGCGCAGAGTTTATGACCCCGGATAAGGCAAAATTACAGCCGTT
GATTGAGGAATTGAAGTTGCATAATCTGCCCATCGGAGGTACGGGAGCCTGCATATCCAATATTGTCCATACCCAGGGCT
GGGTGCACTGCCACAGTGCAGCAACGGATGCTTCAGGACCGGTAAAGGCGATAATGGATGAGCTATACGACTACTTCGTG
TCTATGAAGCTTCCGGCACAGTTGAGAATTTCCTTTGCCTGTTGTCTCAATATGTGTGGGGCTATTCCCTGCTCAGATAT
AGGAATTGTAGGTATACATACCACGGCGCCAAGGGTTGAGCATGAGAAAGTCGGCATACAATGTGAAATTCCGACAACTC
TGGCTAGCTGTCCCACCGGCGCTATCCGCCGGCACCCCGACCCAAACATAAAAAGTGTCGTTATCAACGAAGAACGGTGC
ATGTTTTGCTCTAATTGCTTCACCGTGTGCCCGGCACTGCCGATTGCTGACCCCGAAGGCGACGGACTAGCCGTTTTCGT
TGGCGGTAAAGTCGCCAACGCTAGACGTGGCCCCATGTTTTCCCGGCTGGCTATTCCGTATATTCCTAACACTCCACCTC
GCTGGCCCGAGCTGGTTAACGACGTTAAGAATATTGTTGAAGTTTATGCCGCCAATGCCCGGAAGTGGGAAAGAATGGGC
GAGTGGATAGAGAGGGTAGGCTGGGAGACTTTCTTCAGGCTTACCGGGATACCTTTCACCGACCAGCATATTGATGACTT
CACCCACGCTATAGAAACTTTCAGGACAACCACCCAGTTCAGGTGGTGAGGAAAAGAGGTTGATAATGAGTGAGTTAAGT
GGTGAGGAGCTTGAGCAAAAGATTCTGAAGTATTTGGCAACTGTGGAGATGACTAAAAACAAAAATGTTGCCAGAGCAAT
TGGTGTGGAAAAGAGTCTGGTGGACAAGGCGATAGGCAAACTGGCCATAGAAGACAAGATTGAGTATCTTTACTTGGGCA
CGTCATTTATAAAACTCAAGGGTAAATAACGGGAATACTACAAATAGTGCATAGTGAATCAAAAACTTTCGCCAACCAGT
GGCCGCCCCGTGTTGTCATTGCTGCCCCTCAGGGTCGCTCTGGAAAGACGATTATCAGTGTCGGACTATGCGCTGCTTTC
AAGCGGCGCGGCTTTGTAATTCAATCGTTCAAAAAAGGCCCTGACTATATTGACCCTTCATGGCTTACCGCCGCCGCAGG
CAGAAGCTGTCGCAGTTTGGATGCTGTCTTGATGCCTGAAAGGACATTATTGGCTTCCTTCCAGCAGGCATGCCAGGGGG
CTGACCTGGCTCTTATTGAGGGAGCAATGGGTCTGTATGACAGCTTTGATTCAACAGGCCGGGGGAGTACTGCCCATATA
TCCAGGCTATTGCGTAGTCCTGTTATTCTGGTAATCAATACCGCCCGTATGACACGTAGTATAGCGGCAATGGTAATCGG
GTACCAGCGTTTCGAGCCAGAGACCGATATCGCTGGGGTAATTCTGAATAATGTGGCGGGAAGCCGGCATGAGCACAAAC
TGGTAACCGCCATAGAGCAATACTGCGGGATACCCGTTGTCGGAAGCATACCAAAAGACCTCAGCCTGAGTATCACTGAG
CGACACTTCGGTTTGATACCTTACCGGGAGGCTGAACAGGAGACTGTAATCGACAATATTTGCCATCGTCTTGAAGAGAC
TCTCGACCTGGATAGTATTTTTAATATCGCCCGTGGTGCCAAAGGCAAACGTGTAGTTGCTGCTGCCGCCGAAAGTAAAG
CGGCTATGGTCAAATTAGGAGTAATGCTTGACCGCGTCTTTACGTTCTATTACCCGGAAAATCTTGAAGCGCTGACCCGG
GCCGGCGCCGAACTGGTGTTTATCGACTCTATGCGAGACCAACAGTTACCTGATATTGACGGTCTTTATATCGGTGGCGG
CTTCCCTGAGCTATTTTTACCAGAATTGGAAGCCAATAGCCGACTCCGCCAGCATATTGCTCAGGCAGTCGAAGACTATT
TGCCGGTATATGCTGAGTGTGCCGGTCTCATGTATTTATGCCGGAGTATAAGCTGGCGCGGCGAACAGCATGATATGGTT
GGCGTAATCCCGGCTGAAGTGGAAATATGCCAGCAGCCTCAGGCTCATGGCTATGTAATGGCCGAGGTCGCTGATGAAAA
CCCGTTGTTCCCCATCGGCTTGACTTTTTGGGGCCATGAATTCCATTATTCAAGACTGACCAAATCAAATGACCTCAAAT
TTGCCTATCGGGTGCAACGTGGCCAGGGGATAGATGGCAGAGCAGACGGCATCGTATATAAGAATGTTCTCGCTGCCTAC
ACCCACCTGCATGCTTTAGGTGTGCCCCAGTGGGCAGGCGCTTTCGTACCCCTGGCGTTACGGGAACGGAAACGCCGGGC
CTCATTTTCAGCACTCAAAAATTAAAGGAGGTAGATGATGGCTGAAGAAGGAATGGTCAAGTGTCCAGTGTGTGGTATCG
AGGTACAGGTGGAGTCTGAGGCAAAACTCTACGGGGCGGTAAGGTCTGCTCCAAGGGCTACCAGCACCTATGAAGGTAAA
GACTACTACTTCTGTAGCAAAGCTTGCAAGAAAAAATTCGATGAGTCCCCAGAAAAATTTATTAAGAAATAGGAGGTAAC
ACATGGCAAAAGCTTTACTTGGAGGAATGGAGCTCGAAGTTGATGAGGATGGCTT
132
Supplement
>Aarhus_clone_285
GATTACACACTGGAATCATGGCGGTATAGTTGGGGTCAGGGGCTATGGTGGTGGTGTTATCGGCAGGTATTCGGATGTCG
CTGAAAAGTTCCCAAACGTGGCTGCCTTTCACACCATGAGGGTGAACATGCCCAGCGGCTGGTTCTACTCCACCAAAGCT
TTAAGGAAACTCTGTGATGTCTGGGAAAAGTATGGCAGTGGACTGACCAACTTTCACGGAGCCACGGGTGATATCATACT
TTTAGGGACGACCACTGAGAATCTGCAACCGTGCTTTGATGCCCTGAGTGAAGAGGCGGGATTTGATTTGGGTGGTTCGG
GCTCGGTCTTAAGGACACCAAGCTGCTGCCTCGGACCAGCCAGGTGCGAATGGTCCTGTATAGATACCCTTGATTTATGT
AATGACCTGACTCATGCCTTCCAGGACCAGCTGCATCGGCCTATGTGGCCATACAAGTTTAAGATTAAGATTTCCGGCTG
CCCTAATGACTGTGTTGCCGCTATCGCCCGGTCTGACATGCCGATTATCGGAACCTGGCGTGATGTACTGAGGATAGACC
AAGAAGAGGCAAGGCACTATGTAAACAATGGCTTTGATATCCAGAATGAAGTTATCAACCAGTGTCCTACCCATGCCTTG
GAATGGGATGAGAAAACGCAACAGCTCAAACTGAGAGCTGAGGATTGTGTCAGGTGTATGCACTGTATTAATAGGATGCC
CAGGGCACTAAGGCCAGGGGTGGAAAGAGGGGCGACAATCCTTATCGGTGGTAAAGCTCCGATAATAAAAGGTGCCTACC
TTTCCTGGGTGCTGGTGCCTTTCATGAAGATGGAACCACCTTATGAAGAATTGAAGGAGCTGCTTGAAAAAATATGGGAC
TGGTGGGATGAGCACGGCAGAACGAGGGAAAGAGTGGGTGAGCTCATCGATAGGCTTGGTCTGGCGCGCTTCCTGCGTGA
TGTTGGGCTGAAGCCCGTACCTCAGATGGTATTTCGGCCCCGGTCTAACCCTTACGTTTTCTGGCCATCCGTCGAAGATT
AGACAGGTAAGGCTGACAGGTACCTAAATTTAGGAGATAGTGATAATTATGGCGATAGATATTGGTCCTCCAGACTATAG
AACTATGCTTCACCCGGTAATTAAGAATAACTATGGGAAATGGGACTATCATGAGATATTGCAGCCCGGTGTACTCAGGC
ATGTCTCTGAGAGCGGTGACGAGGTATATACGGTTAGGGCTGGTTCACCCAGACTGTTAAGCGTCGATTCTGTACGGGAG
ATTTGTGATATCGCTGACAAATACTGTGATGGATATATGAGGTTTACCAGCCGCCATTGCATTGAGTTTATGACTCCAGA
TAAAGAAAAGTTACCGTCATTGATTGATGAGATAAAAGCACACAACCTGCCCATCGGTGGTACTGGACGTTCCATATCCA
ATATCGTGCATACTCAAGGTTGGGTACACTGCCACAGCTCGGCAACGGATGCCTCCGGGCCGGTAAAGGCGATAATGGAT
GAATTAATTGACTATTTCACATCTATGAAACTACCGGCTCAGTTGAGAATCTCCTTCGCCTGCTGCCTGAACATGTGCGG
AGCAATTCACTGTTCCGACCTGGCCATTGTGGGTATACATCGGAAACCGCCCAAGGTGGAGCATGAGAGATTAGCCACGG
TGTGTGAAATACCGACGACTCTGGCATCCTGCCCAACCGGGGCTATCCGGCGGCATCCCGACCCGAATGTCAAAAGTGTT
GTGGTTAATGAGGAGCGGTGCATGTACTGTGCCAATTGCTTCACCGTATGTCCGGCAATGCCTATTGCTGACCCGGAAAA
CGATGGCCTCTCTATTTACGTTGGAGGCAAAGTCTCCAATGCCAGGAGTGCCCCTATGTTTTCTCGACTGGCTGTACCCT
TCATCCATAACAATCCACCTCGTTGGCCCGAGCTGGTTGCCGCGGTTAGGAATATTGTGGAAGTCTATGCTGCCAACGCA
CGTAAATGGGAAAGAATGGGTGAGTGGATAGAAAGGGTCGGTTGGGAGAGTTTCTTCAAGTTAACGGGAATACCCTTCAC
CGAACAGCATATCGATGACTTTACTCACGCTGTTGCGACCTTCCGGACTTCGACGCAGTTCAGGTGGTGAAAGTAGAGGT
AGAGATAGTGAACGAACTAAGTAGTGAAGAGCTTGAACAAAAGATTCTTGATTACTTGGCAACAGTGGACATGGCAAAGA
ATCGGAACGTGGCTAAGGCAATTGGTGTGGAAAAGCATTTAGTCGATAAGGCGATAGGTGAATTGGCAAAAGCAGATAAG
CTGGAGTATATTTACCTTGGCACATCATTTGTAAAACTCAAGGGTAAGTAGCAGGACAGGCGATGATGAACAATCCAAAA
GCCTCTGCCACCCGGTGGCCGCCTCGCATTGTAATCGCTGCTCCTCAAGGTCGTTCCGGCAAGACGGTTATTAGTATAGT
TCTATGTGCTAGCCTCAAAAAGCATGGTTTGGTGGTGCAGCCGTTCAAAAAGGGCCCGGACTACATTGACCCTTCTTGGC
TTGCCGTAGCAGGGGGCAGAGACTGCTATAGCCTAGACTCCCGGTTAATACAGGAGCACATGTTACTGGCTTCCTTCCAG
CGGGCTTGCCAGGGGGCAGACTTAGCCCTGATTGAGGGAGCTATGGGACTCTATGACAGCTTTGATTCAACAGGTATGGG
GAGTACTGCCCGTGTAGCCAGACTGTTACAGAGTCCGGTAATTCTAGTAGTTAATTCGGCACGGATTACAAATAGCATTG
CGGCTATGGTACTTGGATACCAGCATTTTGACCCCGAGACAAATATTGCGGCCGTAATCCTGAATAATGTTTCCGGGAGT
AGACATGAACATAAGCTGGTAGCTGCTATAGAGCAGCATTGCGGAATACCGGTTGTTGGGAGTGTACCGAAAGACCGGAA
TCTTAATATCACTGAGCGTCACCTGGGTTTGGTCCCCTACAGAGAAGCTGGAGCTTGGTCGATGGTCGACCGTATCGGTA
ATGGTCTAAATGATAGCCTAAACCTGGAGGCTATTCTGAATATCGCCAGCAGTGCCGGAGATGGCAGCCCGGTGGATGTC
CCATCCCCTGAACGTAGGGAACCGGTAGTTAAAATTGGAGTAATGCTTGACAGCATCTTCACATTCTATTACCCCGAAAA
TCTTGGGGCGCTTGAGAAGGCTGGCGCCACACTGGTGTACATAAACTCCCTGCATGACCAAAAGTTACCTGATATTGACG
GGCTGTACATCGGCGGTGGTTTTCCCGAGCTATTTTTACCCGAACTGGAAGCCAATACCGGGCTACGGAAGCATATAGCG
CAGGCTATTGAGGATGGACTGCCAGTTTATGCCGAGTGTGCTGGCCTAATGTACCTATAATCGAATTCCCGCGGCCGCCA
TGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTC
133
Supplement
>Aarhus_clone_271
GATTGATACACACTGGAATCACGGTGGGGTAGTTGGGGTGAGAGGCTACGGGGGTGGTGTTATTGGCAGGTATTCTGACT
CTCCCGAGAAGTTCCCCAATGCAGCTGCCTTTCACACCTTCAGGGTGAATCAGCCCAGTGGTTGGTTCTACACCACAAAA
GCATTGAGGCAGGTTTGTGATATCTGGGAAAGGTATGGAAGTGGTCTCACCAATTTCCATGGTGCCACCGGTGATATTAT
ACTCCTAGGAACAACGACAGAGAATCTACAGCCTTGCTTTGATGCCCTGACTGGTGAGGCGGGGTTTGATTTAGGAGGGT
CAGCATCAGTATTACGGACACCAAGTGCCTGCGTGGGACCGGCGAGATGTGAATTTGCCTGCATAGATACCCTTGACATA
TGTAATGACCTGACCCATACCTACCAAGATGAGATACATAGACCTCGTTGGCCATATAAATTCAAGTTTAAGATTTCCGG
ATGCCCCAATGATGGTGTCGCCGCTATAGCCAGGGCTGATATGCCTATAATTGGAACCTGGCGAGATGCTCTCAGAATAG
ACCAGGATGCCGTCAGGGAATATGTTGATAATGGCTTTGACATTGTCGGTGAAGTCGTTAGCCAATGCCCCACCCAAGCA
CTGGAGTGGGATGAGAAAGCGAAAAAACTCAACCTGAAGCCAGAGGAATGCGTCAGGTGTATGCATTGTATCAATCAGAT
GCCGAAAGCGCTGAGACCTGGACTGGTCAAGGGGGCTACCATCATGATTGGTGGTAAAGCCCCGCTGGTCAGAGGTGCCC
GTCTCGGCTGGGTTTTAATTCCCTTTATGAAGATGGAACCGCCATACACTGAGCTAAAAGAGTTGGTAGAAAAGATATGG
GATTGGTGGGACGAAAACGCTAGAACCAGGGAACGGGTTTCCGAGCTTATTGAAAGGTTAGGCATGCGTGAGTTCCTCAA
GGCTATAGACCTAGCACCCTGCCCCGAGATGGTAATGGAACCCAGGACTAATGCCTTCATTTTCTAATCGCCAGAGGAAA
TAAACTAAAAGGAGAGTTAAAAAGTGGCTACTGATATAGGACCTCCTGATTACAGGACGATGCTTCCTGAGGTAATAAAG
AAAAACTACGGTAAGTGGAAGTACCACGAAATACTGAAACCTGGCTTATTAAAGCATGTCGCCGAGGGTGGTGATGAACT
CTACACTGTAAGAGCCGGTTCACCCAGATTGTTGAGTATTGATACAGTGCGTGATTTTTGTGATATAGCTGATAAATATT
GCGATGGACACCTGAGATTTACCAGCCGACACAATATAGAGTTTCTGACCGCAGATAAGAGCAAAGTAGAACCACTGATA
CAAGAAGTAAGCAAGCTGGGCTTCCCCGTAGGTGGCACCGGAACCTGCATAAGCAACATTGTTCATACCCAGGGTTGGGT
GCACTGCCATACTCCAGCCACAGATGCCTCCGGAGTGGTGAAAGCCATAATGGATGAGCTATATGACTACTTTGTAGAGG
AGAAGCTCCCGGCTAAGTTCAGGATTTCCCTGGCTTGCTGTGGTAATATGTGTGGCGCCATTCACTGCTCAGATATAGGC
ATTGTTGGCATGCACACCAAACCACCAACAAGGATAGACGATAAGAACTTCATGACCTGGTGCGAGGTTCCAACCACTTT
ATCCTCTTGCCCTACCGGCGCTATCAGGCGATACACTACTGAGGACGGAACGAAAACCGTTAGAATTGACGAAGAGAAGT
GTATGTATTGCGGCAACTGCTATACCGTATGTGCCGCCCTGCCCATAGCTGATGCCGAAGGCGATGGTATAGCCATCTTT
GTTGGGGGCAAGGTTTCCAATGCTAGAACTCGCCCCAAGTTCTCTAAGTTAGCTATCCCCTGGCTTCCCAATAACCCGCC
ACGGTGGCCCGAGGTCGTCAGCGCTGTTAAGAATATTATTGAGGTCTACGCCAGCCATGCCCGAAAGGGGGAGAGGATGG
GCGACTGGATAGAGAGGGTAGGCTGGCCAACCTTCTTCCGATTAACCGGTATACCATTCACCGAGAAACATATTGACGAT
TTTACCGGAGCTTTATCAACCTACAGAACAACTACACAGTTCAAGTGGTAACCGAAGGGGGTGATGATATTGGATGAGAC
ATCAATTGATGAGCTTGAGCAAAAAATATTGAAATACTTGGACACAGTTACCAAAGTAAAGGCACGGGCTGTCCACACGG
CAGTAGGTGAGGACAAAAAACTAGTTGATAAAGCCATGGGCAAATTAGCTAGAGAAGGCAAGGTTGAGTACCTTTACCTG
GAAACATCATATGTAAAACTTGCGGGGAAAGAAATACCAGGTGCTGAGCGGAAGGCTATATAGTCAATCAGAGACTCACT
CTGGCGAGCGGCTGCCTCGGGTTGTTATTTCTGCTTGCCAAGGTCATCTGGGAAAAACAACGGTCAGTATAGGGTTATGT
GCTGCTTATGTTGAACGCGGTCTGGTAGTACAACCCTTTAAGAAGGGTCCCGATTACATCGACCCTTCCTGGCTTACGGC
AGCAGCCAATAGAACCTGTCGTAATCTGGATGCCTACCAGATGCCCGAGGAAGCTATCTTGCTTTCCTTTCAACGTGCCT
CTCTAGGGGCAGACCTAGCTCTTATTGAGGGCGTGATGGGACTTTATGATAGCTTTTCCCCCGATGGCTGGGGCAGCACC
GCCTGGATGGCTCGCCTGCTGGGAGCTCCAGTCATCCTGCTGGTTAATGCCCGCCGAATGACGCGTAGCGTAGCAGCAAT
GATAACCGGCTATCAGCACTTTGAACCGGATACCAATATCGCCGGCGTAATACTGAATAATGTCTTAACTGAGGGGCATA
AGCGAAGGTTGCTGGCAGCTATAGCCCGATATTGTAATGTGCCTGTTCTGGGATGTATACCTCCCGATGATAACCTCAGT
CTTGCTGAGCAGCATCTGGGACTGAGACCCCACCGACACCGGCACTCCGAGGAGACAACCGCTCTTATTGAGCGCATCCG
TGATAGGATTAAATCCTGTCTGGACATAGATGGCATTCTGGCTATCGCCAATAAAGGCGAAACGCAGCGGATACCGGTTG
CAGGTGAGCCGGAGAGAAAAGCACCACTGGTCAAAATAGGAGTATTACGCGACCAGGTTTTCAATTTCTATTACCCGGAA
AACCTGGAGGCATTATGTCAGGCTGGTGCTGAGCTAATCTTCATTAACTCTTTACAAGACCGGAAATTACCTGACATTGA
CGGGCTCTATATGGGTGGTGGCTTCCCTGAATTATTCCTTGAAGGGCTGGAGGCTAATGCCAGCCTGCGCCAGGATATCG
CTCAGGCAGCTAAGAATGGACTACCCATATATGCTGAGTGCGCTGGCCTTATGTACCTCTGTAAAAGCATCTATTGGCGT
GACCGGCGATACGAAATGGTAGGAGCGATTCCTCGCCAGATAGAGATGTGCCAGCGTCCCCAAGGACATGGCTATGTGGA
GGTGGAGGTTGTGGCTGAGAATCCGTTCTTTCCCGTGGGAATGAAAATACGCGGTCATGAATACCATCATTCCAGACTGG
ACAACCTGGATGGTCTTACCTTTGCTTACAAGATGCGGCGGGGTCGGGGAGCAGGTAGGCAGTCAGACGCTATTGTATAT
AATAATGTCTTCGCTACCTACACCCACTTGCACGCTCTAGGTATACCACAATGGGCTGAGGCTTTTGTGTCCTTAGCATC
ACGAGAAAAAAGCCTTAGTCCTGCTTTGTGACGATTAGTTTTGAGGAGGTGAATTTAATGGCAAAAGCTGTACTTGGGGG
CATAGAGATTGAGCTTGATGAGGATGGCTT
134
Supplement
>Aarhus_clone_265
GATTACACACTGGAATCATGGTGGTATAGTCGGGGTCAGGGGCTATGGGGGTGGCGTTATCGGCAGGTATTCTGATGTCC
CGGAAAAGTTCCCAAACGTGGCTGCCTTTCACACCCTTAGAGTGAACATGCCCAGCGGCTGGTTCTACTCTACCAAAGCT
CTAAGGAAGCTCTGCGATGTCTGGGAAAAATATGGCAGTGGACTGACCAACTTTCACGGAGCTACCGGTGATATCATACT
CTTAGGAACGGACACCGAGAATCTGCAACCGTGCTTTGATGCCCTGAGTGATGAAGCGGGGTTTGACTTGGGTGGTTCGG
GCTCGGTCTTGAGGACACCGAGCTGCTGCCTCGGCCCAGCCAGATGCGAATGGTCCTGTATAGACACTCTTGACTTATGT
AACGACCTGACGCACACCTTCCAGGACCAGCTACATCGGCCTATGTGGCCATACAAGTTTAAAATAAAGATTTCCGGCTG
CCCTAATGACTGTGTCGCCTCTATTGCCCGGTCCGACTTGCCCATTATCGGAACCTGGCGCGATGCCCTGAGGATAGACC
AGGGGGAAGTGCGGCAATACGTGAAAAATGGCTTTGATATCCAGAATGAAGTTGTCTCCAGGTGTCCTACCAATGCCCTG
AACTGGGATGAGAAAACACAAGAGCTTGAACTTAGATTTGAAGATTGTGTCAGATGTATGCACTGTATCAACAGGATGCC
CAAGGCACTTAGGCCGGGGATTGAAAGAGGTGCCACCATCCTCATTGGTGGTAAAGCGCCGATAATAAAAGGTGCCTACC
TCTCCTGGGTGCTGGTGCCTTTCATGAAGATGGAGCCACCTTACGATGAATTAAAGGAATTGCTTGAAAAAATATGGGAC
TGGTGGGATGAGCATGGCAGAACCAGGGAAAGGGTTGGTGAGCTCATTAGCAGACTGGGTATGGCACGATTCCTGCGTGA
TATTGGGCTGAAACCCGCACCCCAAATGGTATTTCGACCCAGGTCCAATCCTTATATTTTCTGGCCATCTGATGAAGATA
GAGCAGGTGAAACTGATTGACATATAAATTCAGGAGATAAATATGACGATAGATATTGGTCCTCCAGATTATAGAACCAT
GCTTCACCCGGTGATTAAGAAAAATTATGGGAAATGGCAATATCATGAGAGATTACAGCCTGGTGTACTCAGGCATGTAT
CTGAGTCCGGTGATGAGGTATTTACGATTAGAGTTGGTTCACCCAGGCTGTTAAGTGTCGATTCGATACGGGAAATTTGT
GACATTGCTGATAAACACTGTGATGGCTATCTGAGGTTTACCAGCCGCCATTGCATTGAATTTATGACCCCGGATAAGAA
CAAGTTGGAGTCATTAATTGGGGAGATAAAAGCGTACAATCTACCCATCGGTGGCACGGGGCGTTCCATATCTAATATTG
TACATACTCAGGGTTGGGCGCACTGCCATAGCTCGGCAACAGATGCCTCCGGACCGGTAAAAGCAATAATGGACGAATTA
ATTGATTATTTCACATCGGATAAGCTACCGGCTCTATTGAGAATATCCTTCGCCTGCTGCCTGAACATGTGCGGAGCGAT
TCATTGTTCGGACATTGCCATTGTGGGTATACATACGAAGCCTCCCAAGGTAGATGACCAGAGACTGGCCACTGTTTGTG
AAATACCAACCACCCTGGCATCCTGCCCTACGGGGGCTATACGGCGACATCCTGACCCGAATGTAAAAAGTGTGGTGGTT
AATGAGGAACGGTGCATGTACTGTGCCAATTGCTTCACCGTATGTCCGGCCATGCCTATTGCCGACGCCGAAAATGATGG
ACTTTCTATTTATGTTGGCGGCAAGGTCTCCAATGCCAGAAGTGCTCCCATGTTCTCTCGACTGGCGGTACCCTTCATCC
CTAACAAACCGCCTCGCTGGCCCGAGCTGGTTAAGGAAGTGAAAAATATCGTGGAAGTCTATGCGGCTCATGCCCGTAAA
TGGGAAAGAATGGGCGAATGGATAGAAAGGGTCGGCTGGGAGACTTTCTTTAAGTTAACAGGAATACCTTTCACAGAACA
ACATATCGATGATTTCACCCATGCCGTGACAACCTTCCGGAGTTCGACACAGTTTCGGTGGTGAAAGTAGAGGATAAGAT
AGTGAACGAGCTAAATAATGAAGAGCTTGAACAAAAGATTCTCAAATACCTGGCAACAGTGAACATGGCCAAGAACCGAA
ATGTGGCTAAGGCGATTGATGTGGAAAAGAAGCTGGTTGATAAGGCAATAGGCAAACTGGCCAAGGAAGATAAGCTGGAG
TACCTTTACCTGGGCACCTCATTTGTCAAAATCAAGGGTAAGTAGCATAAAAGACAGTGATGAACGATTCAAAAGTCTTG
GCTACCACATGGCCACCTCGTATTATAATCGCTGCTCCTCAAGGTCATTCAGGGAAGACGGTTATTAGTATCGGTCTCTG
CGCCAGCCTCAAACAGCGTGGCTTGGTGGTTCAGCCGTTTAAAAAGGGGCCAGACTACATTGACCCTTCCTGGTTGGCCG
CGGCCGGGGGTAGAGACTGCCATAACCTGGACCCCCTATTAATGCCGGAGCAATCATTACTGGCTTCCTTCGAGCGGGCT
TGCCAGGGAGCAGGCCTGGCCCTCATTGAAGGATCCATGGGACTCTATGACAGCCTCGATACAACCGGTCAGGGAAGTAC
TGCCCGCGTAGCCAGACTATTACAGAGTCCAGTAATTTTGATAGTTAATTCGGCACGGATGACTAATAGCATAGCCGCTA
TGGTACTTGGATACCAGCACTTTGACACGGAGACGAATATTGCGGCCGTAATTCTCAATAATGTTTCCGGGAGCCGGCAT
GAGCAGAAACTGGTGGCGGCTGTAGAGCAGCATTGTGGAATACCAGTGGTAGGGAGCGTACCGAGAGATGCCACTCTCGA
TATTGCGGAGCGTCACCTTGGTTTAATTCCCTACAGGGAGGCTGGAGACTGGTCTATTATCGAACGCATCGGTAATTGCC
TCAAAAACAACCTTAACCTGGAGGCTATCCTTGCTATCGCCCACAATGCTGGAGGTGGCAGCCCGGTGAATGTCCCATTC
CCTAAACGTAAGAAAACTGTGGTTAGAATCGGGGTAATGCTTGACCACGTCTTTACCTTCTATTATCCGGAAAATTTTGC
GGCACTTATACAGGCCGGCGCAGAACTGGTCTTTATCAACTCCGTACATGACCATAGATTGCCCGATATTGACGGGTTGT
ACATCGGTGGCGGTTTCCCTGAGCTATATTTGTCGGAATTGGAAGCCAATATATCGATGCGACAGCATATAGCCAAAGCC
ATTAATAATGGGCTGCCGGTCTATGCTGAGTGTGCCGGCCTGATGTATTTATGCCGGGGCATTAGGTGGCACAGCAGGTG
GCATGAAATGGTAGGAGTGATACCCGCTAATGTGGAAATAGGTCAAAAGCCTAAAGGTCACGGCTATGTATCGGCTGAGG
TTACTCAGGAGAACCCCTGGTTACCGGTTGGCTTGAAATTTTGGGGGCATGAGTTCCATTATTCCAAGATGACCATATCT
GGTGAGCTTAACTTTGCCTATCAGATAGGGCGCGGGTATGGCATCGATGGCAGGAAAGATGGTGTCATATATAAAAATAT
GCTGGGTGCTTACACACACCTGCATAGTCTTGGTGTCTCCCATTGGGCGGATGCCTTCGTATCTCTAGCCTTACGAGAGC
GCAAAGGTCACCGCTCGCTTTCAGTACTGACACATTAATCTGGCGAACGCTACCGTGCCAGCATAAAGGAAAGGAGAGGA
TTTATGCCGAAAGCAACACTTGGTGGACAGGAAATAGAGCTTGATGAGGATGGCTT
135
Supplement
>Aarhus_clone_252
GATTACACACTGGAATCATGGCGGTGTAGTTGGTGTGAGGGGTTATGGAGGTGGGGTTATCGGCAGATATTCTGATTCCC
CTGAGAAGTTTCCTAATGTAGCTGCCTTTCACACGTTTAGGGTAAATCAGCCAAGCGGTTGGTTCTACACTACTAAAGCC
TTAAGGCAGATTTGTGATATTTGGGAAAGATACGGAAGTGGGCTGACGAACTTTCATGGTGCAACGGGTGATGCTATACT
CCTGGGAGCACCCACTGAGAATTTGCAGTCTTGCTTCGATGCCCTGACTGATGAGGCCGGGTTTGATTTAGGCGGCTCAG
CCTCAGTCTTGCGGACGCCAAGTGCTTGTATGGGACCAGCCAGATGTGAATATGCCTGTATAGACACCCTTGATATATGC
AATGACCTGACCCATACCTTCCAAGACTATATACATAGACCTCACTGGGCATATAAATTCAAGATTAAGGTTTCCGGGTG
TCCTAATGACTGTGTTGCTGCTATAGCCCGAGCCGATATGCCAATAATCGGTACCTGGAGGGGTGCTCTTAGGATAGACC
AGGATGCGGTCAGGGAATATGTTGATAGTGGATTTGACATTAAAGGCGAGGTCGTTAGCCAGTGTCCTAGCGAAGCGCTG
GAGTGGGATGAAAAAGCGAAGAGGCTTAACCTGAAACCAGAGGAGTGCGTCAGATGCATGCATTGCATAAACAAGATGCC
TAAAGCGCTAAGACCAGGGCTGGACAAGGGAGCTACCATCATGATTGGCGGTAAGGCTCCACTGGTGAGAGGTGCCCGCC
TTGGTTGGGTATTAGTTCCTTTTATGAAAATGGAGCCACCATATACTGAGTTGAAAGAATTACTGGAAAAGATATGGGAT
TGGTGGGACGAAAATGCCAGGACTAGGGAAAGGATTTCCGAGCTTATCGAGAGGTTAGGGCTGCGAGAGTTCCTCAAGGC
TATAGACTTGCCACCCTGCCCTGAGATGGTATTGGAACCGAGGGCTAATGCCTTCATTTTCTAGCGACCTGAGGAATCTA
GAATAAACTGAAAGGAACGTAGGATATGAGCAGTGATATAGGACCTCCAGATTATAGAACAATGCTTCCTGAAGTAATGA
AGAAGAATTATGGGAAGTGGAAGTATCACGAGATATTGAGACCTGGCGTATTAAAGCATGTGGCCGAAGGTGGGGATGAA
ATCTATACTGTAAGGGCAGGTTCACCTAGGTTGCTGAGCATTGACACTTTGCGCGATATCTGCGATGTAGCTGATAAATA
TTGTGACGGGCACCTTAGATTTACCAGCCGAAACAATATAGAATTTCTGTTCACTGACCAGAGTAAGATAGAGCCATTAA
CAGAGGAGATAAAAAATAAGCTAGGGTTCCCCATAGGCGGCACTGGAACCTGCATAACCAATATTGTTCACACTCAAGGT
TGGGTACACTGTCATACTCCAGCCACAGACGCCTCAGGTGTGGTGAAAGCCATAATGGATGAGCTATATGATTACTTTGT
GTCGGAGAAGCTCCCGGCTAAGTTGAGGATTTCCGAGGCTTGCTGTGGTAATATGTGTGGTGCCATTCACTGCTCTGATA
TAGGTATTGTGGGGGTACATACCAAACCACCAACAAAGATAGATGATAAGAACTTCCCCACCTGGTGCGAGGTTCCAACC
ACCCTAGCCAGTTGCCCGACCGGTGCCATCAGGCGATACCCGGAGGCAAAAAGCGTCAGGGTTGATGAAGAGAAGTGTAT
GTATTGCGGCAACTGCTACACCGTATGTGCTGCTATGCCAATTGCCGATGCCGAAGGCGATGGCATCTCCATCTTTGTTG
GGGGCAAGGTCTCCAATGCTAAAACTCGCCCCAGGTTCTCCAGATTAGCTATTCCCTGGATACCGAACAACCCGCCAAGA
TGGCCTGAGGTTGTCAGCGCCGTTAAGAATATTATTGAGGTGTACGCTAACCATGCCCGAAAAGGAGAAAGGATGGGCGA
TTGGATAGAGAGAGTAAGTTGGCCAACTTTCTTCCGCTTAACCGGTATACCATTTACTGAGAAACATATTGATGATTTTA
CCGGGGCTCTGTCAAGCTATAGGACAACAACACAATTTAAGTTGTAAAGGGGTGATTATATTGGCTGACACGGTTAGTGA
TGAGCTTGAGCAAAAAATATTAAAATACCTAGAAACGGTTAGCATGGTAAAGGTACGAGCTGTTCACAAGTCAATCAATG
AGGACAAAAAGCTGGTTGACCAGGCAATAGGCAAACTAGCTAAAGAGGACAAGATTGAGTACCTTTACTTGGATACATCG
TATGTGAAGCTTAAGGAGAGGCAGCGGTAACAAATTGTAATGGTGAGTAGTGAATCACAGGCTCATAGTAGTGGGAGCCT
ACCTCAGATTGTTATTTCTGCCTGTCAAGGTCATTCGGGCAAGACTACTGTCAGTGTAGGGTTATGTGCTGCTTGGGCTG
AACGGGGCTTGATTGTGCAGCCTTTCAAAAAGGGGGCGGATTATATCGACCCTTCTTGGCTTACAGCAGCAAGCAACAGG
GCTTGCCGTAACTTAGATGCCTTCCTAATGCCTAAAGAAACCGTCTTACGCTCCTTTCAGCGTGCTTGCCAGAGGACGGA
CCTGGCTCTTATTGAGGGGGTGATGGGGCTATATGACAGCTATGATACTGGTGGTGAGGGGAGCACGGCGTGGGTGGCTC
GCCTGCTGGGAGCGCCGGTCATTCTGCTGATAAACGCCAGTCGGATGACACGTAGCGTGGCAGCTATGGTAACAGGTTAT
CAGCGCTTTGAGCCAGATACGTGGATTGCTGGTGTCATATTGAACAATGTTTCGTTAAGCGGTCACAGAAGGAAGCTGAT
GGCGGCTATAGAGCGATATTGTGATATACCTGTGTTGGGGAGCATACCCAGTGACAGCAGTTTCGCAATTGCTGAGCAGC
ACCTGGGCTTAAGACCATACAAGGCAACCGAAGATGCACCATCTATCATCAAGCGCCTCCGTGATAATATTAAGGGCTAC
CTTGATTTGGACGGGATCTTAGCAATCGCTCGAAAAGGTGAAGCGCAGCTGATGCCAGGGGTAAGTGAGACCCAGAGAGA
GGCACCGTTAGTCCAGATAGGGGTAATGTTTGATAGGGCTTTTAATTTTTATTACCCGGAGAACCTGGAGGCCTTATACC
AGGCTGGTGCTAACTTGGTCTTTATCGATTCCCTGCAAGACCAGAGGCTACCTGATATTGACGGGCTCTATATTGGGGGG
GGGTTCCCCGAGTTATTCCCCAGGGAACTGGAAGCCAATAGCCAGCTTCGCCAAGATGTTGCTCAAGCTGTCAAAGGTGG
CCTACCGGTATATGCCGAGTGCGCTGGACTTATGTACCTGTGTCAGGGTATACGTTGGCAGCACCAGCGATACGAAATGG
TTGGCGTAATTCCCAGCGAAGTAGAGATGTGCTCTCAGACGCAGGGGCATGGCTATGTGGTGGCTGAGGTGACTAGGGAA
AACCCCTGGCTACCTGTTGGTTTAACTGTCCGTGGCCACCAATTCCATTTTTCCCGACTCTCTAACCCAAGTGACCTTGA
TTTTGCCTATCGTATGAAACGAAGGCGGGAGAATGATCCCAAAGTGGATGGCATTGTATATAAGAATCTGTTTGCCGCTT
ATACCCACCTGCATACCTATAGTGTCCCCCTGTGGGCAGAGTCGTTCGTATCGCTGGCATTACGAGAGCGAAAATGTCGG
CTCCCACCTTCGACACTAACAACCTGAGTGTCAAATGGCTGACACGCTTTCATACTTGGGGGAGAAGACAAGGAACATTA
GAGCCTACAGAGTTAAAAAGGAAAGGGGATAAACACATGCCAAAGACCACTCTTGGTGGAATAGAGTTTGAAGTTGATGA
GGATGGCTT
136
Supplement
>Aarhus_clone_215
GATTACGCACTGGAAACATGGTGGTATAGTCGGGGTCAGGGGCTATGGGGGTGGCGTTATCGGCAGGTATTCTGATGTCC
CGGAAAAGTTCCCAAACGTGGCTGCCTTTCACACCCTTAGAGTGAACATGCCCAGCGGCTGGTTCTACTCTACCAAAGCT
CTAAGGAAGCTCTGCGATGTCTGGGAAAAATATGGCAGTGGACTGACCAACTTTCACGGAGCTACCGGTGATATCATACT
CTTAGGAACGGACACCGAGAATCTGCAACCGTGCTTTGATGCCCTGAGTGATGAAGCGGGGTTTGACTTGGGTGGTTCGG
GCTCGGTCTTGAGGACACCGAGCTGCTGCCTCGGCCCAGCCAGATGCGAATGGTCCTGTATAGACACTCTTGACTTATGT
AACGACCTGACGCACACCTTCCAAGACCAGCTACATCGGCCTATGTGGCCATACAAGTTTAAAATAAAGATTTCCGGCTG
CCCTAATGACTGTGTCGCCTCTATTGCCCGGTCTGACTTGCCCATTATCGGAACCTGGCGCGATGCCCTGAGGATAGACC
AGGGGGAAGTGCGGCAATACGTGAAAAATGGCTTTGATATCCAGAATGAAGTTGTCTCCAGGTGTCCTACCAATGCCCTG
AACTGGGATGAGAAAACACAAGAGCTTGAACTAAGATTTGAAGATTGTGTCAGATGTATGCACTGTATCAACAGGATGCC
CAAGGCACTTAGGCCGGGGATTGAAAGAGGTGCCACCGTCCTCATTGGTGGTAAAGCGCCGATAATAAAAGGTGCCTACC
TCTCCTGGGTGCTGGTGCCTTTCATGAAGATGGAGCCACCTTACGATGAATTAAAGGAATTGCTTGAAAAAATATGGGAC
TGGTGGGATGAGCATGGCAGAACCAGGGAAAGGGTTGGTGAGCTCATTAGCCGACTGGGTATGGCACGATTCCTGCGTGA
TATTGGGCTGAAACCCGCACCCCAAATGATATTTCGACCCAGGTCCAATCCTTATATTTTCTGGCCATCTGATGAAGATA
GAGCAGGCGAAACTGATTGACATATAAATTCAGGAGATAAATATGACGATAGATATTGGTCCTCCAGATTATAGAACCAT
GCTTCACCCGGTAATTAAGAAAAATTATGGGAAATGGCAATATCATGAGAGATTACAGCCTGGTGTACTCAGGCATGTAT
CTGAGTCCGGTGATGAGGTATTTACGGTTAGAGTTGGTTCACCCAGGCTGTTAAGTGTCGATTCGATACGGGAAATTTGT
GATATTGCTGATAAACACTGTGATGGCTATCTGAGGTTTACCAGCCGCCATTGCATTGAATTTATGACCCCTGATAAGAA
CAAGTTGGAGCCATTAATTGGGGAGATAAAAGCATACAACCTACCCATCGGTGGCACGGGGCGTTCCATATCTAATATTG
TACATACTCAGGGTTGGGCGCACTGCCATAGCTCGGCAACAGATGCCTCCGGACCGGTAAAAGCAATAATGGACGAATTA
ATTGATTATTTCACATCGGATAAGCTACCGGCTCTATTGAGAATATCCTTCGCCTGTTGCCTGAACATGTGCGGAGCGAT
TCATTGTTCGGACATTGCCATTGTAGGTATACATACGAAGCCTCCCAAGGTAGATGACCAGAGACTGGCCACTGTTTGTG
AAATACCAACCACACTGGCATCCTGCCCTACGGGGGCTATACGGCGACATCCTGACCCGAATGTAAAAAGTGTGGTGGTT
AATGAGGAACGGTGCATGTACTGTGCCAATTGCTTCACCGTATGTCCGGCCATGCCTATTGCCGACGCCGAAAATGATGG
GCTTTCCATTTATGTTGGCGGCAAGGTCTCCAATGCCAGAAGTGCTCCCATGTTCTCTCGACTGGCGGTACCCTTCATCC
CTAACAAACCGCCTCGCTGGCCCGAGCTGGTTAAGGAAGTGAAAAATATCGTGGAAGTCTATGCGGCTCATGCCCGTAAA
TGGGAAAGAATGGGCGAATGGATAGAAAGGGTCGGCTGGGAGACTTTCTTTAAGTTAACAGGAATACCTTTCACAGAACA
ACATATCGATGATTTCACCCATGCCGTGACAACCTTCCGGAGTTCAACACAGTTTCGGTGGTGAAAGTAGAGGATAAGAT
AGTGAACGAGCTAAATAATGAAGAGCTTGAACAAAAGATTCTCAAATACCTGGCAACAGTGAACATGGCCAAGAACCGAA
ATGTGGCTAACGCGATTGGTGTGGAAAAGAAGCTGGTTGATAAGGCAATAGGCAAACTGGCCAAGGAAGATAAGCTGGAG
TACCTTTACCTGGGCACCTCATTTGTCAAAATCAAGGGTAAGTAGCATAAAAGACAGTGATGAACGATTCAATAGCCTCG
ACTATCACATGGCCACCTCGTATCATAATCGCTGCTCCTCAAGGTCATTCCGGGAAGACGGTTATTAGTATCGGTCTCTG
TGCCAGCCTCAAACAGCGTGGCTTGGTGGTTCAGCCGTTTAAAAAGGGGCCAGACTACATTGACCCTTCCTGGTTGGCCG
CGGCCGGGGGTAGAGACTGCCATAACCTGGACCCCGTATTAATACCGGAGCAATCATTACTGGCTTCCTTCGAGCGGGCG
TGCCAGGGGGCAGGCCTGGCCCTCATTGAAGGATCCATGGGACTCTATGACAGCCTCGATACAACCGGTCAGGGAAGTAC
TGCCCACGTAGCCAGACTATTACAGAGTCCAGTAATTTTGATAGTTAATTCGGCACGGATGACTAATAGCATAGCCGCTA
TGGTACTTGGATACCAGCACTTTGACACGGAGACGAATATTGCGGCCGTAATTCTCAATAATGTTTCCGGGAGCCGGCAC
GAGCAGAAACTGGTGGCGGCTGTAGAGCAGCATTGTGGAATACCAGTGGTAGGGAGCGTACCGAGAGATGCCACTCTCGA
TATTGCGGAGCGTCACCTTGGTTTAATTCCCTACAGGGAGGCTGGAGACTGGTCTATTATCGAACGCATCGGTAATTGCC
TCAAAAACAACCTTAATCTGGAGGCTATCCTTGCTATCGCCCACAATGCTGGAGGTGGCAGCCCGGTGAATGTCCCATTC
CCTAAACGTAAGAAAACTGTGGTTAGAATCGGGGTAATGCTTGACCACGTCTTTACCTTCTATTATCCGGAAAATTTTGC
GGCACTTATACAGGCCGGCGCAGAACTGGTCTTTATCAACTCCGTACATGGCAATAGATTGCCCGATATTGACGGGTTGT
ACATCGGTGGCGGTTTCCCTGAGCTATATTTGTCGGAATTGGAAGCCAATATATCGATGCGACAGCATATAGCCAAAGCC
ATTAATAATGGGCTGCCGGTCTATGCTGAGTGTGCCGGCCTGATGTATTTATGCCGGGGCATTAGGTGGCACAGCAGGTG
GCATGAAATGGTAGGAGTGATACCCGCTAATGTGGAAATAGGTCAAAAGCCTAAAGGTCACGGCTATGTATCGGCTGAGG
TTACTCAGGAGAACCCCTGGTTACCGGTTGGCTTGAAATTTTGGGGGCATGAGTTCCATTATTCCAAGATGACCATATCT
GGTGAGCTTAACTTTGCCTATCAGATAGGGCGCGGGTATGGCATCGATGGCAGGAAAGATGGTGTCATATATAAAAATAT
GCTGGGTGCTTACACACACCTGCATAGTCTTGGTGTCTCCCATTGGGCGGATGCCTTCGTATCTCTAGCCTTACGAGAGC
GCAAAGGTCACCGCTCGCTTTCAGTACTGACACATTAATCTGGCGAACGCTACCGTGCCAGCATAAAGGAAAGGAGAGGA
TTTATGCCGAAAGCAACGCTTGGTGGACAGGAAATAGAGGTTGATGAGGATGGCTT
137
Supplement
>Aarhus_clone_G28
TATCGGGAGGTATTCTGACTCTCCCGAGAAGTTCCCCAATGCAGAAGCCTTCCATACCTTCAGGGTAAATCAACCAAGCG
GGTGGTTCTACACCACCAAAGCCTTAAGGCAGGTTTGTGATATCTGGGAAAAGTATGGAAGCGGACTCACCAATTTGCAC
GGGGCAACCGGTGATATGATACTCTTGGGAACAACGACAGAGAATCTGCAGGCTTGCTTTGATGCCCTGAGCGGTGAGGC
GGGGTTTGACCTGGGAGGGTCAGCGTCAGTCCTGAGGACACCAAGTGCTTGCGTGGGACCGGCGAGATGCGAATTTGCCT
GCATAGATACCCTTGACATATGTAATGACCTGACTCAGACCTACCAAAACTATATACACCGACCCCACTGGGCATATAAA
TTCAAGATTAAGATTTCTGGATGTCCCAACGATGGTGTCGCTGCTATAGCCAGGTCTGATATGCCCATAATCGGAACCTG
GCGGGATGCTCTCAGAATAGACCAGGATGCCGTCAGGGAATATGTTGACAATGGCTTTGACATTGTCGGTGAGGTCGTTA
GCCAATGCCCCACTGAAGCACTGGAATGGGATGCCAAAGTGAAAAAGCTCAACCTTAAGCCAGAAGAATGTGTCAGGTGT
ATGCATTGTATCAATAAGATGCCGAAAGCGCTAAGACCTGGACTGGACAAGGGGGCTACCATTCTGATTGGTGGTAAAGC
CCCGCTGGTCAGAGGTGCCCGTCTCGGCTGGGTATTAGTACCCTTTATGAAGATGGAACCGCCATACACTGAGCTAAAAG
AGCTGATAGAAAAGATATGGGATTGGTGGGATGAAAACGCTAGAACCAGGGAACGGATTGCCGAGCTTATTGAAAGGTTA
GGCATGCGTGAGTTCCTCAAGGCTATAGACCTAGCACCCTGCCCTGAGATGGTAATGGAACCCAGGGCTAATGCTTTCAT
TTTCTAGTCATAAGAAAAGTGAACTGGAAGGAGTAGAAAATTGGGCACGGATATAGGACCTCCAGACTATAGAACAATGC
TTCCTGAGGTAATAAAGAAAAACTACGGTAAGTGGAAGTATCATGAGATACTGAAACCTGGCTTATTAAAGCATGTCGCC
GAGGGTGGTGATGAACTCTACACTGTAAGAGCCGGTTCCCCCAGATTGCTGAGTATTGATACTATACGCGATTTCTGTGA
CATAGCTGATAAATATTGCAATGGACACCTGAGATTTACCAGCCGACACAATATAGAGTTTCTGACCGCAGATAAGAGCA
AAGTGGAGCCACTGATACAAGAAGTAAGCAAGCTGGGCTTCCCCGTGGGTGGTACCGGAAAGTGCATAAGCAATATTGTT
CATACGCAGGGCTGGGTGCACTGTCACACTCCAGCCACAGATGCCTCCGGAGTGGTGAAAGCCATAATGGATGAGCTATA
TGACTACTTTGTGTCA
>Aarhus_clone_H16
GTTATCGGGAGGTATTCTGACTCTCCCGAGAAGTTCCCCAATGCAGCCGCCTTCCATACCTTCAGGGTAAATCAACCAAG
CGGGTGGTTCTACACCACCAAAGCCTTAAGGCAGGTTTGTGATATCTGGGAAAAGTATGGAAGCGGACTCACCAATTTGC
ACGGGGCAACCGGTGATATGATACTCCTGGGAACAACGACAGAGAATCTGCAGGCTTGCTTTGATGCCCTGAGCGGTGAG
GCGGGGTTTGACCTGGGAGGGTCAGCGTCAGTCCTGAGGACACCAAGTGCTTGCGTGGGACCGGCGAGATGCGAATTTGC
CTGCATAGATACCCTTGACCTGTGTAATGACCTGACTCAGACCTACCAAAACTATATACACCGACCCCACTGGGCATATA
AATTCAAGATTAAGATTTCCGGATGTCCCAATGATGGTGTCGCCGCTATAGCCAGGTCTGATATGCCCATAATCGGAACC
TGGCGAGATGCTCTCAGAATAGACCAGGATGCCGTCAGGGAATATGTTGACAATGGCTTTGACATTGTCGGTGAGGTCGT
TAGCCAATGCCCCACTGAAGCACTGGAATGGGATGCCAAAGCGAAAAAGCTCAACCTTAAGCCAGAAGAATGTGTCAGGT
GTATGCATTGTATAAATAAGATGCCGAAAGCGCTAAGACCTGGACTGGACAAGGGGGCTACCATTCTGATTGGTGGTAAA
GCCCCGCTGGTCAGAGGTGCCCGTCTCGGCTGGGTATTAGTACCCTTTATGAAGATGGAACCGCCATACACCGAGCTAAA
AGAGCTGATAGAAAAGATATGGGATTGGTGGGATGAAAACGCTAGAACCAGGGAACGGATTGCCGAGCTTATTGAAAGGT
TAGGTATGCGTGAGTTCCTCAAGGCTATAGACCTAGCACCCTGCCCTGAGATGGTAATGGAACCCAGGGCTAATGCTTTC
ATTTTCTAGTCATAAGAAAAGTGAACTGGAAGGAGTAGAAAATTGGGCACGGATATAGGACCTCCAGACTATAGAACAAT
GCTTCCTGAGGTAATAAAGAAAAACTACGGTAAGTGGAAGTATCATGAGATACTGAAACCTGGCTTATTAAAGCATGTCG
CCGAGGGTGGTGATGAACTCTACACTGTAAGAGCCGGTTCCCCCAGATTGCTGAGTATTGATACTATACGCGATTTCTGT
GACATAGCTGATAAATATTGCAATGGACACCTGAGATTTACCAGCCGACACAATATAGAGTTTCTGACCGCAGATAAGAG
CAAAGTGGAGCCACTGATAC
>Aarhus_clone_H20
GGGGGTGGTGTTATCGGGAGGTATTCTGACTCTCCCGAGAAGTTCCCCAATGCAGCCGCCTTCCATACCTTCAGGGTAAA
TCAACCAAGCGGGTGGTTCTACACCACCAAAGCCTTAAGGCAGGTTTGTGATATCTGGGAAAAGTATGGAAGCGGACTCA
CCAATTTGCACGGGGCAACCGGTGATATGATACTCCTGGGAACAACGACAGAGAATCTGCAGGCTTGCTTTGATGCCCTG
AGCGGTGAGGCGGGGTTTGACCTGGGAGGGTCAGCGTCAGTCCTGAGGACACCAAGTGCTTGCGTGGGACCGGCGAGATG
CGAATTTGCCTGCATAGATACCCTTGACCTGTGTAATGACCTGACTCAGACCTACCAAAACTATATACACCGACCCCACT
GGGCATATAAATTCAAGATTAAGATTTCCGGATGTCCCAATGATGGTGTCGCCGCTATAGCCAGGTCTGATATGCCCATA
ATCGGAACCTGGCGAGATGCTCTCAGAATAGACCAGGATGCCGTCAGGGAATATGTTGACAATGGCTTTGACATTGTCGG
TGAGGTCGTTAGCCAATGCCCCACTGAAGCACTGGAATGGGATGCCAAAGCGAAAAAGCTCAACCTTAAGCCAGAAGAAT
GTGTCAGGTGTATGCATTGTATAAATAAGATGCCGAAAGCGCTAAGACCTGGACTGGACAAGGGGGCTACCATTCTGATT
GGTGGTAAAGCCCCGCTGGTCAGAGGTGCCCGTCTCGGCTGGGTATTAGTACCCTTTATGAAGATGGAACCGCCATACAC
CGAGCTAAAAGAGCTGATAGAAAAGATATGGGATTGGTGGGATGAAAACGCTAGAACCAGGGAACGGATTGCCGAGCTTA
TTGAAAGGTTAGGTATGCGTGAGTTCCTCAAGGCTATAGACCTAGCACCCTGCCCTGAGATGGTAATGGAACCCAGGGCT
AATGCTTTCATTTTCTAGTCATAAGAAAAGTGAACTGGAAGGAGTAGAAAATTGGGCACGGATATAGGACCTCCAGACTA
TAGAACAATGCTTCCTGAGGTAATAAAGAAAAACTACGGTAAGTGGAAGTATCATGAGATACTGAAACCTGGCTTATTAA
AGCATGTCGCCGAGGGTGGTGATGAACTCTACACTGTAAGAGCCGGTTCCCCCAGATTGCTGAGTATTGATACTATACGC
GATTTCTGTGACATAGCTGATAAATATTGCAATGGGACACCTGGAGATTTACCAGCCCGACACAATATAGAAGTTTCTGA
CCGCAGATAAGAG
138
Supplement
>Aarhus_clone_75
GTAAAGCAATTGGCACAGTACATGCACCGTTCCTCATTAACCACCACACTCTTTACATTCGGGTCAGGATGTCGCCGTAT
AGCCCCCGTAGGGCAGGATGCCAGTGTGGTTGGTATTTCACAGACAGTGGCCAGCTTCTGGTCATCTACCTTGGGAGGCT
TCGTGTGTATACCCACAATGGCAATATCCGAACAATGAATCGCTCCGCACATGTTCAGGCAGCAGGCGAAGGATATTCTC
AACATAGCCGGCAGCTTATCCGATGTGAAATAATCAATTAATTCGTCCATTATTGCTTTTACCGGTCCGGAGGCATCTGT
TGCCGAGCTATGGCAGTGCGCCCAACCCTGAGTATGTACAATATTGGATATGGAACGTCCCGTGCCACCGATGGGCAGGT
TGTGAGCTTTTATCTCCCCAATTAATGGCTCTAACTTGTTCTTATCCGGGGTCATAAATTCAATGCAATGGCGGCTGGTA
AACCTCAGATAGCCATCACAGTGTTTATCAGCAATATCACAAATTTCCCGTATCGAATCGACACTTAACAGCCTGGGTGA
GCCAACCCTAACCGTAAATACCTCATCACCGGACTCAGATACATGCCTGAGTACACCAGGCTGTAATCTCTCATGATATT
GCCATTTCCCGTAATTTTTTTTAATTACCGGGTGAAGCATGTTTCTATAATCTGGAGGGCCAATATCTATCGTCATATTT
ATCTCCTGAATTTATACGTCAATCAGTTTTACCTGCTCTATCTTCATCAGATGGCCAGAAAATATAAGGATTGGACCTGG
GTCGAAATACCATTTGGGGTGCGGGTTTCAGCCCAATATCACGCAGGAATCGTGCCATACCCAGTCTGCTAATGAGCTCA
CCAACCCTTTCCCTGGTTCTGCCATGCTCATCCCACCAGTCCCATATTTTTTCAAGCAATTCCTTTAATTCATCGTAAGG
TGGCTCCATCTTCATGAAAGGCACCAGCACCCAGGAGAGGTAGGCACCTTTTATTATCGGCGCTTTACCACCAATGAGGA
CGGTGGCACCTCTTTCAATCCCCGGCCTAAGTGCCTTGGGCATCCTGTTGATACAGTGCATACATCTGACACAATCCTCA
AATCTTAGTTCAAGCTCTTGTGTTTTCTCATCCCAGTTCAGGGCATTGGTAGGGCACCTGGAGACAACTTCATTCTGGAT
ATCAAAGCCATTTTTAACGTATTGCCGCACTTCCTCCTGGTCTATCCTCAGGGCATCGCGCCAAGTTCCGATAATGGACA
AGTCGGACCGGGCGATAGAGGCGACACAGTCATTAGGGCAGCCGGAAATCTTTATTTTAAACTTGTATGGCCACATAGGC
CGATGTAGCTGGTCCTGGAAGGTATGCGTCAGGTCGTTACATAAGTCAAGAGTGTCTATACAGGACCATTCGCATCTGGC
TGGGCCGAGGCAGCAGCTTGGTGTCCTCAAGACCGAGCCCGAACCACCCAAGTCAAACCCCGCTTCATCACTCAGAGCAT
CAAAGCACGGTTGCAGATTCTCGGTGTCCGTTCCTAAGAGTATGATATCACCGGTGGCTCCGTGAAAGTTGGTCAGTCCA
CTGCCATATTTTTCCCAGACATCGCAGAGTTTCCTTAGAGCTTCAGTAGAGTAGAACCAGCCGCTGGGCATGTTCACTCT
AAGGGTGTGAAAGGCAGCCACGTTTGGGAACTTTTCGGGGACATCAGAATACCTGCCGATAACACCACCCCCATAGCCCC
TGACCCCGACTATACCACCATGTTTCCAGTGTGTAATCCTATCCTCATAGGAAAGCTCCAGTTGCCCAAGAAGGTCCTTA
GCTGCCGGTTTTCTCTTCGCTACCTTTTTAATTTCCGTCACAAAGCTTGGCCAAGGCCCGCCCTCAAGGTCATCGAGCTG
TGGAGTTTCACTTTTTGTCATATAACACCCCCGTATAATTTTAGTAGAAAAATGTGATCAATAGGTAGAACTTCTGAAAT
GGTTTTTGGCCCTATTTATATTAGATTGAGCTCGACACTCTCATCAAGGTAATTCTAGCACTAACAGGTAGTTTATTCAA
TCAACTCACTCTTGGGTAATATTTATCAGATTGTTTTCTGTCAATTTCGATGGTCGTGTAGTGGTATAATTCAAGCTGTG
AGCGGGATGATGTGAAATATGGAAGGATAATAGGTTTTAAGGTTTAGTCAGTACATAGGAGCATATTTAATGGACAGAAG
GAAAAAACTTGTCAATGAATACAAGCAGCGCAAACAAAGTGGTGGCGTATACAAGATAACCAACAACCTGAGCGGGAAGT
ATCTCCTGGACCGTGCCTATGACCTTAGAGGGATGCAAAGCCGCTTTAATTTCTCGGTTTCCACTGGCTCTTGTATTCAT
CCCCGATTGCAGGAAGATTGGAAGGAATTCGGAGGTAAGGTGTTTACCTTTGAAATACTTGAGTCAATCGAAAAAAGGAA
GACCAGAGCCAAGATGAGTTTACGGATGACCTGAAAACACTTGAGGAAATATGGTGCGGGAAACTCGATGCATCAAACGA
ATACTGAGCCGGATGAAAAGTTCTAGAGAATCGAATTCCCGCGGCCGCCATGGCGGCCGGGAGCATGCGACG
139
Supplement
>Aarhus_clone_20
GTAAAGCAATTGGCACAGTACATGCACCGTTCCTCATTAACCACCACACTCTTTACATTCGGGTCAGGATGTCGCCGTAT
AGCCCCCGTAGGGCAGGATGCCAGTGTGGTTGGTATTTCACAGACAGTGGCCAGCTTCTGGTCATCTACCTTGGGAGGCT
TCGTGTGTATACCCACAATGGCAATATCCGAACAATGAATCGCTCCGCACATGTTCAGGCAGCAGGCGAAGGATATTCTC
AACATAGCCGGCAGCTTATCCGATGTGAAATAATCAATTAATTCGTCCATTATTGCTTTTACCGGTCCGGAGGCATCTGT
TGCCGAGCTATGGCAGTGCGCCCAACCCTGAGTATGTACAATATTGGATATGGAACGTCCCGTGCCACCGATGGGCAGGT
TGTGAGCTTTTATCTCCCCAATTAATGGCTCTAACTTGTTCTTATCCGGGGTCATAAATTCAATGCAATGGCGGCTGGTA
AACCTCAGATAGCCATCACAGTGTTTATCAGCAATATCACAAATTTCCCGTATCGAATCGACACTTAACAGCCTGGGGGA
GCCAACCCTAACCGTAAATACCTCATCACCGGACTCAGATACATGCCTGAGTACACCAGGCTGTAATCTCTCATGATATT
GCCATTTCCCGTAATTTTTTTTAATTACCGGGTGAAGCATGTTTCTATAATCTGGAGGACCAATATCTATCGTCATATTT
ATCTCCTGAATTTATATGTCAATCAGTTTTACCTGCTCTATCTTCATCAGATGGCCAAAAAATATAAGGATTGGACCTGG
GTCGAAATACCATTTGAGGTGCGGGTTTCAGCCCAATATCACGCAGGAATCGTGCCATACCCAGTCTGCTAATGAGCTCA
CCAACCCTTTCCCTGGTTCTGCCATGCTCATCCCACCAGTCCCATATTTTTTCAAGCAATTCCTTTAATTCATCGTAAGG
TGGCTCCATCTTCATGAAAGGCACCAGCACCCAGGAGAGGTAGGCACCTTTTATTATCGGCGCTTTACCACCAATGAGGA
TGGTGGCACCTCTTTCAATCCCCGGCCTAAGTGCCTTGGGCATCCTGTTGATACAGTGCATACATCTGACACAATCCTCA
AATCTTAGTTCAAGCTCTTGTGTTTTCTCATCCCAGTTCAGGGCATTGGTAGGGCACCTGGAGACAACTTCATTCTGGAT
ATCAAAGCCGTTTTTCACGTATTGCCGCACTTCCTCCTGGTCTATCCTCAGGGCATCGCGCCAAGTTCCGATAATGGGCA
AGTCGGACCGGGCGATAGAGGCGACACAGTCATTAGGGCAGCCGTAAATCTTTATTTTAAACTTGTATGGCCACATAGGC
CGATGTAGCTGGTCCTGGAAGGTATGCGTCAGGTCGTTACATAAGTCAAGAGTGTCTATACAGGACCATTCGCATCTGGC
TGGGCCGAGGCAGCAGCTTGGTGTCCTCAAGACCGAGCCCGAACCACCCAAGTCAAACCCCGCTTCATCACTCAGAGCAT
CAAAGCACGGTTGCAGATTCTCAGTGTCCGTTCCTAAGAGTATGATATCACCGGTGGCTCCGTGAAAGTTGGTCAGTCCA
CTGCCATATTTTTCCCAGACATCGCAGAGCTTCCTTAGAGCTTCGGTAGAGTAGAACCAGCCGCTGGGCATGTTCACTCT
AAGGGTGTGAAAGGCAGCCACGTTTGGGAACTTTTCGGGGACATCAGAATACCTGCCGATAACACCACCCCCATAGCCCC
TGACCCCGACTATACCACCATGTTTCCAGTGTGTAATCCTATCCTTATAGGAAAGCTCCAGTTGCCCAAGAAGTTCCTTA
GCTGCCGGTTTTCTCTTCGCTACCTTTTTAATTTCCGTCACAAAGCTTGGTCAAGGCCCGCCCTCAAGGTCATCAAGCTG
TGGAGTTTCACTGTTTGCCATATAACACCCCCGGATAATTATAGTAGAAATATGTGATCAATAGGTAGAACTTCTGAAAT
GGTTTTTGGCCCTATTTATATTAGATTAAGCTCGACAACTCTCATCAAGGTAATTCTAGCACTAACAGGTAGTTTGTTCA
ATCAACTCACTCTTGGGTACTATATATCAGATTGTTTTCTGTCAATTTCGATGGTCGTGTAGTGGTATAATTCAAGCTGT
GAGCGGGATGATGTGAAATATGGAAGGATAATAGGTTTTAAGGTTTAGTCAGTACATAGGAGCATATTTAATGGACAGAA
GGAAAAAACTTGTCAATGAATACAAGCAGCACAAACAAAGTGGTGGTATATACAAGATAACCAACAACCTGAGCGGGAAG
TATCTCCTGGGCCGTGCCTATGACCTTAGAGGGATGCAAAACCGCTTTAATTTCTCGGTTTCCACTGGCTCTTGTATTCA
TCCCCGGTTGCAGGAAGATTGGAAGGAATTCAGAGGTAAGGTGTTTACCTTTGAAATACTTGAGTCAATCGAAATAAAAG
AAGACCAGAGCCAAGATAAGTTCACGGATGACCTGAAAACACTTGAGGAAATATGGCGCGGGAAACTCGATGCATCAAAC
GAATACTGAGCCGGATGAAAAGTTCTAGAGAATCGAATTCCCGCGGCCGCCATGGCGGCCGGGAGCATGCGACGTCGGGC
CCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTT
ACCCAACT
140
Supplement
>Aarhus_clone_11
GTAAAGCAATTGGCACAGTACATGCACCGTTCCTCATTAACCACCACACTTTTTACATTCGGGTCAGGATGTCGCCGTAT
GGCCCCCGTAGGGCAGGATGCCAGTGTGGTTGGTATTTCACAGACAGTGGCCAGTCTCTCATCATCTACCTTGGGTGGCT
TCGTATGTATACCCACAATGGCAATGTCCGAACAATGAATCGCTCCGCACATGTTCAGGCAGCAGGCGAAGGATATTCTC
AACATAGCTGGCAGCTTATCCGCTGTGAAATAATCGATTAACTCGTCCATTATTGCTTTTACCGGTCCTGAGGCATCTGT
TGCCGCGCTATGACAATGTGCCCAACCCTGAGTATGTACAATATTGGATATGGAACGTCCCGTGCCACCGATGGGCAGGT
TGTGAGCTTTTATCTCCCCAATTAATGGCTCTAACTTGTTCTTATCCGGGGTCATAAATTCAATGCAATGGCGGCTGGTA
AACCTCAGATAGCCATCACAGTGTTTATCAGCAATATCACAAATTTCCCGTATCGAATCGACACTTAACAGCCTGGGGGA
GCCAACCCTAACCGTAAATACCTCATCACCGGACTCAGATACATGCCTGAGTACACCAGGCTGTAATCTCTCATGATATT
GCCATTTCCCGTAATTTTTTTTAATTACCGGGTGAAGCATGTTTCTATAATCTGGAGGACCAATATCTATCGTCATATTT
ATCTCCTGAATTTATATGTCAATCAGTTTTACCTGCTCTATCTTCATCAGATGGCCAAAAAATATAAGGATTGGACCTGG
GTCGAAATACCATTTGAGGTGCGGGTTTCAGCCCAATATCACGCAGGAATCGTGCCATACCCAGTCTGCTAATGAGCTCA
CCAACCCTTTCCCTGGTTCTGCCATGCTCATCCCACCAGTCCCATATTTTTTCAAGCAATTCCTTTAATTCATCGTAAGG
TGGCTCCATCTTCATGAAAGGCACCAGCACCCAGGAGAGGTAGGCACCTTTTATTATCGGCGCTTTACCACCAATGAGGA
CGGTGGCACCTCTTTCAATCCCCGGCCTAAGTGCCTTGGGCATCCTGTTGATACAGTGCATACATCTGACACAATCCTCA
AATCTTAGTTCAAGCTCTTGTGTTTTCTCATCCCAGTTCAGGGCATTGGTAGGGCACCTGGAGACAACTTCATTCTGGAT
ATCAAAGCCGTTTTTCACGTATTGCCGCACTTCCTCCTGGTCTATCCTCAGGGCATCGCGCCAAGTTCCGATAATGGGCA
AGTCGGACCGGGCGATAGAGGCGACACAGTCATTAGGGCAGCCGGAAATCTTTATTTTAAACTTGTATGGCCACATAGGC
CGATGTAGCTGGTCCTGGAAGGTATGCGTCAGGTCGTTACATAAGTCAAGAGTGTCTATACAGGACCATTCGCATCTGGC
TGGGCCGAGGCAGCAGCTTGGTGTCCTCAAGACCGAGCCCGAACCACCCAAGTCAAACCCCGCTTCATCACTCAGAGCAT
CAAAGCACGGTTGCAGATTCTCAGTGTCCGTTCCTAAGAGTATGATATCACCGGTGGCTCCGTGAAAGTTGGTCAGTCCA
CTGCCATATTTTTCCCAGACATCGCAGAGCTTCCTTAGAGCTTCGGTAGAGTAGAACCAGCCGCTGGGCATGTTCACTCT
AAGGGTGTGAAAGGCAGCCACGTTTGGGAACTTTTCGGGGACATCAGAATACCTGCCGATAACACCACCCCCATAGCCCC
TGACCCCGACTATACCACCATGTTTCCAGTGTGTAATCCTATCCTTATAGGAAAGCTCCAGTTGCCCAAGAAGTTCCTTA
GCTGCCGGTTTTCTTTTCGCTACCTTTTTAATTTCCGTCACAAAGCTTGGCCAAGGCCCGCCCTCAAGGTCATCAAGCTG
TGGAGTTTCACTGTTTGCCATATAACACCCCCGGATAATTATAGTAGAAATATGTGATCAATAGGTAGAACTTCTGAAAT
GGTTTTTGGCCCTATTTATATTAGATTAAGCTCGACACTCTCATCAAGGTAATTCTAGCACTAACAGGTAGTTTGTTCAA
TCAACTCACTCTTGGGTACTATATATCAGATTGTTTTCTGTCAATTTCGATGGTCGTGTAGTGGTATAATTCAAGCTGTG
AGCGGGATGATGTGAAATATGGAAGGATAATAGGTTTTAAGGTTTAGTCAGTACATAGGAGCATATTTAATGGACAGAAG
GAAAAAACTTGTCAATGAATACAAGCAGCACAAACAAAGTGGTGGTATATGCAAGATAACCAACAACCTGAGCGGGAAGT
ATCTCCTGGGCCGTGCCTATGACCTTAGAGGGATGCAAAACCGCTTTAATTTCTCGGTTTCCACTGGCTCTTGTATTCAT
CCCCGGTTGCAGGAAGATTGGAGGGAATTCGGAGGTAAGGTGTTTACCTTTGAAATACTTGAGTCAATCGAAATAAAAGA
AGACCAGAGCCAAGATAAGTTCACGGATGACCTGAAAACACTTGAGGAAATATGGCGCGGGAAACTCGATGCATCAAACG
AATACTGAGCCGGATGAAAAGTTCTAGAGAATCGAATTCCCGCGGCCGCCATGGCGGCCGGGAGCATGCGACGTCGGGCC
CAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTA
CCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCC
CAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC
AGCGTGACCGCTACACTTGCCAGCGCCCTAGCG
141
Supplement
>BaffinBay_clone_1
GATTACACACTGGAAGCACGGTGGTATAGTTGGCGTTAGGGGCTATGGGGGAGGGGTTATAGGTAGGTACTCCGATGTCCCCG
AGCAATTCCCAAATGTAACTGCCTTCCACACTATAAGGGTAAATATGCCCAGTGGCTGGTTCTACACCACCAAGGCTCTTAGA
GGTGTCTGTGATGTCTGGGAAAAATATGGTAGCGGTCTCACTAATCTCCACGGGGCGACAGGTGACATCATACTTTTGGGAAC
GACTTCGGAAAATTTACAGCCTTGTTTTGACGCACTGAGTGATGAGGCGGGATTTGATTTGGGGGGTTCTGGCTCAGTCTTGA
GGACGCCAAGCTGTTGTGTGGGACCGGCTAGATGTGAATGGTCCTGCATAGACACTCTTGACATATGTAACGACCTGACTCAT
GAATTTCAGGACGAGCTACACCGACCCATGTGGCCATATAAATTCAAAATTAAGATATCCGGATGCCCCAATGACTGTGTTGC
CGCCATTGCTAGAGCCGATATGCCCATAATCGGCACCTGGCGTGATTACCTTAGAGTAGACCAGGATGAAGTGAGAAAATATG
TAGCCGGTGGCTTCGATATCCAGAGAGAAGTTATCGCTATGTGTCCCACCTGGGCACTGGACTGGGATGAAAAAGCACAGGAG
CTCAAGGTGAAGCAAGAAGAATGTGTCAGGTGCATGCATTGTATTAACCGAATGCCTAAGGCAATCAGGCCGGGGGTTGAGCG
AGGAGCTACCATTCTTATCGGTGGTAAAGCGCCGATAATAAAGGGGGCACTGCTTTCCTGGGTGCTGGTTCCCTTTATGAAGA
TGGAGCCACCCTATACCGAATTCAAGGAGCTGGCACGCAAAATATGGGAATGGTGGGACGAAAATGGCAGGACAAGGGAAAGG
GTTGGCGAACTCATTGAGAGATTGGGCATGGCGCAGTTCCTGCGAGAAATGGGACTAAAGCCAATCCCTCAAATGGTGTTCCG
ACCCAGGTCCAACCCGTATGTCTTCTGGCCCCCAGAAAAGAGGAGAAAGTAAAGGGAATAAAATAAATCAGTTGAGGAGAGCG
CTTTGACTATTGATATTGGTCCTCCAGATTATAAGCTTATGCTTCACCCGGTAATCAAGGAAAACTATGGCAAGTGGAAATAC
CATGAGATATTAGAGCCAGGTGTGCTGAAGCACGTATCTGAGACCGGGGGCGAGGTATATACAGTACGAGTGGGCTCGCCCAG
GCTGGTAAGCATTGACTTCATACGAGAGATTTGTGATATCGCTGACAAATACTGTGACGGTTACTTAAGGTTTACCAGTCGGC
ATTGCATAGAGTTTATGACCCCTGATGAGGCAAAATTACAGCCGTTGATTGAGGAGTTGAAATCGCATAATCTGCCCATCGGT
GGTACGGGAGCCTGTATATCCAATATTGTCCATACCCAGGGCTGGGTGCACTGCCACAGTGCAGCTACAGATGCCTCAGGACC
GGTAAAGGCGATAATGGATGAGTTATACGACTACTTCGTATCTATGAAGCT
>BaffinBay_clone_14
TTACACACTGGAAGCATGGCGGTATAGTTGGTGTCACCGGGTACGGTGGTGGAGTTATCGGTAGATACTCCGACGTTCCAGAG
GAGTTCCCAAGTGTCGAATCTTTCCACACCTTCCGGGTAAACCAGCCCAGTGGTTGGTTCTACACCACAAAAGCTCTGAGGGA
ACTTTGTGATGTCTGGGAGAAGTATGGCAGTGGTATGACCAACTTTCACGGTTCAACTGGTGACATCATACTTCTGGGAGCAA
CCACTGAGAATTTACAGCCTTGCTTTGACGCATTGACTGAAATAGGCTTTGATTTAGGTGGTTCGGGGGGGGACTTGAGGACG
CCCAGTTGTTGCGTTGGTCCGGCTCGTTGTGAATATGCCTGCATAGATACTCTCGACATATGTCATGACTTGACTATGACATA
CCAAGACGAGATACACCGACCGAGGTGGCCCTATAAATTCAAAATTAAGGTTTCTGGCTGTCCTAACGATTGCGTAGCTGCCA
TAGCCAGGTCCGATTTGACTATAATTGGTACCTGGAAAGATAACCTCAGGATTGACCAGGCCGCCGTCAGGGAGTATGTAGAG
GGCGGGCTTGATATCAAAAACATAGTCATCGACAAATGCCCGACTAAGGCACTGGAATGGGATGAGAAAGCAGCCGAACTTAA
ACTGAGGCTAGAGGATTGCGTCAGGTGCATGCACTGCATAAACAAGATGCCCAAAGCCCTAAGACCAGGGCTTGATAGAGGAG
CCACCATCCTGATTGGTGGCAAAGCACCTATCATCAAAGGCGCTTACCTCTCCTGGGTACTGGTTCCTTTCATGAGGATGGAG
CCTCCATATACCGAAGTCAAGGAGTTGCTGCGTAAAATATGGGATTGGTGGGATGAGAACGGCAGGACAAGAGAAAGGGTGGC
CGAGCTTATTGAAAGAGCAGGGCTAAAGACATTCCTCCGTGCCATTGGGCTAAAGCCAGTGCCACAGATGGTATTTAAACCCA
GAGCAAATCCATATGTTTTCTTCGATTGATTGGGAAGTTAGTAAAAGGAGATCAGCAATGACTAAGACTGATATTGGTCCTCC
AGATTACCGTTTGATGCTTCCAGAAATAATAAAGAAAAACTACGGAAAGTGGAAATATCATGATATATTACAGCCGGGAGTGC
TCAAACATGTATCTGAAACCGGCGATGAAGTCTATACGGTAAGAGCAGGCTCGCCAAAACTGGTAAGTATAGATTTTATCCGC
GACATTTGTGACATAGCTGATAAATACTGCGATGGTCACCTAAGGTTCACCAGCCGATACAACATTGAGTTCATGACTCCCGA
CAAGACCAAGGTAGAACTGATAATTGAAGCGGTTAAAAAGCTTGGTCTTCCCGTGGGTGGTACTGGAAAATCTATATCCAATA
TTGTGCACACCCAGGGCTGGATTCATTGCCATAGCGCGGCTACTGATGCCTCTGGTTTAGTGAAAGCGCTAATGGACGAGCTT
TATGACTACTTTACTACCATGAAACTCCCGGCTAAGCTGAGGATTGCCTTAGCTTGCTGCATAAACATGTGTGGAGCAGCCCA
CTGCTCAGATTTAGCCGTTGTCGGCATCCATACCAAGGTACCAAAGGTAGATGACGAGAGGGTGCCCATCGTCTGTGAAATTC
CAACAACTATGGCCGCCTGTCCTACCGGGGCAATCCGTCGACATCCAGACTCGAATAT
>BaffinBay_clone_22
GGTGGTATAGTTGGCGTTAGGGGCTATGGGGGAGGGGTTATAGGTAGGTACTCCGATGTCCCCGAGCAATTCCCAAATGT
AACTGCCTTCCACACTATAAGGGTAAATATGCCCAGTGGCTGGTTCTACACAACCAAGGCTCTTAGAGGTGTCTGTGATG
TCTGGGAAAAATATGGTAGCGGTCTCACTAATCTCCACGGGGCGACAGGTGACATCATACTTTTGGGAACGACTTCGGAA
AATTTACAGCCTTGTTTTGACGCACTGAGTGATGAGGCGGGATTTGATTTGGGGGGTTCTGGCTCAGTCTTGAGGACGCC
AAGCTGTTGTGTGGGGCCGGCTAGATGTGAATGGTCTTGCATAGACACTCTTGACATATGTAACGACCTGACTCATGAAT
TTCAGGACGAGCTACACCGACCCATGTGGCCATATAAATTCAAAATTAAGATATCTGGATGCCCCAATGACTGTGTTGCC
GCCATTGCTAGAGCCGATATGCCCATAATCGGCACCTGGCGTGATTACCTTAGAGTAGACCAGGATGAAGTGAGAAAATA
TGTAGCCGGTGGATTCGATATCCAGAGAGAAGTTATCGCTATGTGTCCCACCTGGGCACTGGACTGGGATGAAAAAGCAC
AGGAGCTCAAGGTGAAGCAAGAAGAATGTGTCAGGTGCATGCATTGTATTAACCGAATGCCTAAGGCAATCAGGCCGGGG
GTTGAGCGAGGAGCTACCATTCTTATCGGTGGTAAAGCGCCGATAATAAAGGGGGCACTGCTTTCCTGGGTGCTGGTTCC
CTTTATGAAGATGGAGCCACCCTATACCGAATTCAAGGAGCTGGCACGCAAAATATGGGAGTGGTGGGACGAAAATGGCA
GGACGAGGGAAAGGGTTGGCGAACTCATTGAGAGATTGGGCATGGCGCAGTTCCTGCGAGAAATGGGACTAAAGCCAATC
CCTCAAATGGTGTTCCGACCCAGGTCCAACCCGTATGTCTTCTGGCCTCCAGAAAAGAGGAGGAAGTAAAGGGAATAAAA
TGAATCAGTTAAGGAGAGCACTATGACTATTGATATTGGTCCTCCAGATTATAAGCTTATGCTTCACCCGGTAATCAAGG
AAAACTATGGCAAGTGGAAATACCATGAGATATTAGAGCCGGGTGTGCTGAAGCACGTATCTGAGACCGGGGGCGAGGTA
TATACAGTACGAGTGGGCTCGCCCAGGCTGGTAAGCATTGACTTCATACGAGAGATTTGTGATATCGCTGACAAATACTG
TGACGGTTACTTAAGGTTTACCAGTCGGCATTGCATAGAGTTTATGACCCCTGATGAGGCAAAATTACAGCCGTTGATTG
AGGAGTTGAAATCGCATAATCTGCCCATCGGTGGTACGGGAGCCTGTATATCCAATATTGTCCATACCCAGGGCTGGGTG
CACTGCCACAGTGCAGCTACAGATGCCTCAGGACCGGTAAAGGCGATAATGGATGAGTTATACGACTACTTCGTATCTAT
GAAGCTCCCGGCACAGTTGAGAATTTCCTTTGCCTGTTGTCTCAACATGTGTGGAGCTATTCCCTGCTCAGATATAGGAA
142
Supplement
TTGTGGGCATACATACCATGGTGCCAAGGGTGGAGCATGAGAAAGTCGGCATACAATGTGAAATCCCGACGACTCTGGCT
AGCTGTCCCACCGGCGCTATCCGCCGGCACCCCGACCCAAACATAAAAAGTGTCGTTATCAACGAAGAACGGTGCATGTT
TTGCTCTAATTGCTTCACCGTGTGCCCGGCACTGCCGATTGCTGACCCCGAGGGCGACGGACTAGCCATTTTCGTTGGCG
GTAAAGTCGCCAACGCTAGACGTGGCCCCATGTTTTCCCGGCTGGCTATTCCGTATATTCCTAACACTCCACCTCGCTGG
CCCGAGCTGGTTAACGACGTTAAGAATATTGTTGAAGTATATGCCGCCAATGCCCGGAAGTGGGAGAGAATGGGCGAGTG
GATAGAGAGGGTAGGCTGGGAGACTTTCTTCAGGCTTACCGGGATACCTTTCACCGACCAGCATATTGATGACTTCACCC
ACGCTATAGAAACTTTCAGGACAACCACCCAGTTCAGGTGGTGAGGAAAAGAGGTGATAATGAGCGAGTTAAGTGGTGAG
GAGCTCGAGCAAAAGATTCTGAAGTATTTGGCAACTGTGGAGATGACTAAAAACAAAAATGTTGCCAGAGCAATTGGTGT
GGAAAAGAGTCTGGTGGACAAGGCGATAGGCAAACTGGCCATAGAAGACAAGATTGAGTATCTTTACTTGGGCACGTCAT
TTATAAAACTCAAGGGTAAATAACGGGAATACTGCAAATAGTGCATAGTGAATCAAAAACTTTCGCCAACCAGTGGCCGC
CCCGTGTTGTCATTGCTGCCCCTCAGGGTCGCTCCGGAAAGACGATTATCAGT
>BaffinBay_clone_38
GATTACACACTGGAAGCATGGCGGTATAGTTGGTGTCACCGGGTACGGTGGTGGAGTTATCGGTAGATACTCCGACGTTCCAG
AGGAGTTCCCAAGTGTCGAATCTTTCCACACCTTCCGGGTAAACCAGCCCAGTGGTTGGTTCTACACCACAAAAGCTCTGAGG
GAACTTTGTGATGTCTGGGAGAAGTATGGCAGTGGTATGACCAACTTTCACGGTTCAACTGGTGACATCATACTTCTGGGAGC
AACCACTGAGAATTTACAGCCTTGCTTTGACGCATTGACTGAAATAGGCTTTGATTTAGGTGGTTCGGGGGGGGACTTGAGGA
CGCCCAGTTGTTGCGTTGGTCCGGCTCGTTGTGAATATGCCTGCATAGATACTCTCGACATATGTCATGACTTGACTATGACA
TACCAAGACGAGATACACCGACCGAGGTGGCCCTATAAATTCAAAATTAAGGTTTCTGGCTGTCCTAACGATTGCGTAGCTGC
CATAGCCAGGTCCGATTTGACTATAATTGGTACCTGGAAAGATAACCTCAGGATTGACCAGGCCGCCGTCAGGGAGTATGTAG
AGGGCGGGCTTGATATCAAAAACATAGTCATCGACAAATGCCCGACTAAGGCACTGGAATGGGATGAGAAAGCAGCCGAACTT
AAACTGAGGCTAGAGGATTGCGTCAGGTGCATGCACTGCATAAACAAGATGCCCAAAGCCCTAAGACCAGGGCTTGATAGAGG
AGCCACCATCCTGATTGGTGGCAAAGCACCTATCATCAAAGGCGCTTACCTCTCCTGGGTACTGGTTCCTTTCATGAGGATGG
AGCCTCCATATACCGAAGTCAAGGAGTTGCTGCGTAAAATATGGGATTGGTGGGATGAGAACGGCAGGACAAGAGAAAGGGTG
GCCGAGCTTATTGAAAGAGCAGGGCTAAAGACATTCCTCCGTGCCATTGGGCTAAAGCCAGTGCCACAGATGGTATTTAAACC
CAGAGCAAATCCATAGGTTTTCTTCGATTGATTGGGAAGTTAGTAAAAGGAGATCAGCAATGACTAAGACTGATATTGGTCCT
CCAGATTACCGTTTGATGCTTCCAGAAATAATAAAGAAAAACTACGGAAAGTGGAAATATCATGATATATTACAGCCGGGAGT
GCTCAAACATGTATCTGAAACCGGCGATGAAGTCTATACGGTAAGAGCAGGCTCGCCAAAACTGGTAAGTATAGATTTTATCC
GCGACATTTGTGACATAGCTGATAAATACTGCGATGGTCACCTAAGGTTCACCAGCCGATACAACATTGAGTTCATGACTCCC
GACAAGACCAAGGTAGAACTGATAATTGAAGCGGTTAAAAAGCTTGGTCTTCCCGTGGGTGGTACTGGAAAATCTATATCCAA
TATTGTGCACACCCAGGGCTGGATTCATTGCCATAGCGCGGCTACTGATGCCTCTGGTTTAGTGAAAGCGCTAATGGACGAGC
TTTATGACTACTTTACTACCATGAAACTCCCGGCTAAGCTGAGGATTGCCTTAGCTTGCTGCATAAACATGTGTGGAGCAGCC
CACTGCTCAGATTTAGCCATTGTCGGCATCCATACCAAGGTACCAAAGGTAGATGACGAGAGGGTGCCCATCGTCTGTGAAAT
TCCAACAACTATGGCCGCCTGTCCTACCGGGGCAATCCGTCGACATCCAGACTCGAATATAAAAAGCGTTATCGTCAATGAAG
AGCGCTGCATGTACTG
>BaffinBay_clone_48
GATTACACACTGGAAGCATGGTGGTATAGTTGGCGTTAGGGGCTATGGGGGAGGGGTTATAGGTAGGTACTCCGATGTCCCCG
AGCAATTCCCAAATGTAACTGCCTTCCACACTATAAGGGTAAATATGCCCAGTGGCTGGTTCTACACCACCAAGGCTCTTAGA
GGTGTCTGTGATGTCTGGGAAAAATATGGTAGCGGTCTCACTAATCTCCACGGGGCGACAGGTGACATCATACTTTTGGGAAC
GACTTCGGAAAATTTACAGCCTTGTTTTGACGCACTGAGTGATGAGGCGGGATTTGATTTGGGGGGTTCTGGCTCAGTCTTGA
GGACGCCAAGCTGTTGTGTGGGACCGGCTAGATGTGAATGGTCTTGCATAGACACTCTTGACATATGTAACGACCTGACTCAT
GAATTTCAGGACGAGCTACACCGACCCATGTGGCCATATAAATTCAAAATTAAGATATCTGGATGCCCCAATGACTGTGTTGC
CGCCATTGCTAGAGCCGATATGCCCATAATCGGCACCTGGCGTGATTACCTTAGAGTAGACCAGGATGAAGTGAGAAAATATG
TAGCCGGTGGATTCGATATCCAGAGAGAAGTTATCGCTATGTGTCCCACCTGGGCACTGGACTGGGATGAAAAAGCACAGGAG
CTCAAGGTGAAGCAAGAAGAATGTGTCAGGTGCATGCATTGTATTAACCGAATGCCTAAGGCAATCAGGCCGGGGGTTGAGCG
AGGAGCTACCATTCTTATCGGTGGTAAAGCGCCGATAATAAAGGGGGCACTGCTTTCCTGGGTGCTGGTTCCCTTTATGAAGA
TGGAGCCACCCTATACCGAATTCAAGGAGCTGGCACGCAAAATATGGGAGTGGTGGGACGAAAATGGCAGGACGAGGGAAAGG
GTTGGCGAACTCATTGAGAGATTGGGCATGGCGCAGTTCCTGCGAGAAATGGGACTAAAGCCAATCCCTCAAATGGTGTTCCG
ACCCAGGTCCAACCCGTATGTCTTCTGGCCTCCAGAAAAGAGGAGGAAGTAAAGGGAATAAAATGAATCAGTTAAGGAGAGCA
CTATGACTATTGATATTGGTCCTCCAGATTATAAGCTTATGCTTCACCCGGTAATCAAGGAAAACTATGGCAAGTGGAAATAC
CATGAGATATTAGAGCCGGGTGTGCTGAAGCACGTATCTGAGACCGGGGGCGAGGTATATACAGTACGAGTGGGCTCGCCCAG
GCTGGTAAGCATTGACTTCATACGAGAGATTTGTGATATCGCTGACAAATACTGTGACGGTTACTTAAGGTTTACCAGTCGGC
ATTGCATAGAGTTTATGACCCCTGATGAGGCAAAATTACAGCCGTTGATTGAGGAGTTGAAATCGCATAATCTGCCCATCGGT
GGTACGGGAGCCTGTATATCCAATATTGTCCATACCCAGGGCTGGGTGCACTGCCACAGTGCAGCTACAGATGCCTCAGGACC
GGTAAAGGCGATAATGGATGAGTTATACGACTACTTCGTATCTATGAAGCTCCCCGCACAGTTGAGAATTTCCTTTGCCTGTT
GTCTCAACATGTGTGGAGCTATTCCCTGCTCAGATATAGGAATTGTGGGCATACATACCATGGTGCCAAGGGTGGAGCATGAG
AAAGTCGGCATACAATGTGAAATCCCGACGACTCTGGCTAGCTGTCCCACCGGCGCTATCCGTCGGCACCCCGAC
143
Literature
Literature
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Adrian, L. (2009). ERC-group microflex: microbiology of Dehalococcoides-like
Chloroflexi. Rev. Environ. Sci. Biotechnol. 8: 225–229.
Yamada, T., Y. Sekiguchi, S. Hanada, H. Imachi, A. Ohashi, H. Harada, and Y.
Kamagata. (2006). Anaerolinea thermolimosa sp. nov., Levilinea saccharolytica gen.
nov., sp. nov. and Leptolinea tardivitalis gen. nov., sp. nov., novel filamentous
anaerobes, and description of the new classes Anaerolineae classis nov. and
Caldilineae classis nov. in the bacterial phylum Chloroflexi. Int. J. Sys. Evol.
Microbiol. 56: 1331–1340.
Cavaletti, L., P. Monciardini, R. Bamonte, P. Schumann, M. Rohde, M. Sosio,
and S. Donadio. (2006). New lineage of filamentous, spore-forming, gram-positive
bacteria from soil. Appl. Environ. Microbiol. 72: 4360–4369.
Hugenholtz, P., and E. Stackebrandt. (2004). Reclassification of Sphaerobacter
thermophilus from the subclass Sphaerobacteridae in the phylum Actinobacteria to
the class Thermomicrobia (emended description) in the phylum Chloroflexi (emended
description). Int. J. Sys. Evol. Microbiol. 54: 2049–2051.
Gupta, R. S., P. Chander, and S. George. (2013). Phylogenetic framework and
molecular signatures for the class Chloroflexi and its different clades; proposal for
division of the class Chloroflexi class. nov. into the suborder Chloroflexineae subord.
nov., consisting of the emended family Oscillochloridaceae and the family
Chloroflexaceae fam. nov., and the suborder Roseiflexineae subord. nov., containing
the family Roseiflexaceae fam. nov. Antonie Van Leeuwenhoek 103: 99–119.
Löffler, F. E., J. Yan, K. M. Ritalahti, L. Adrian, E. A. Edwards, K. T.
Konstantinidis, J. A. Müller, H. Fullerton, et al. (2013). Dehalococcoides mccartyi
gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to
halogen cycling and bioremediation, belong to a novel bacterial class,
Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family
Dehalococcoidaceae fam. nov., within the phylum Chloroflexi. Int. J. Sys. Evol.
Microbiol. 63: 625–635.
Adrian, L., U. Szewzyk, J. Wecke, and H. Görisch. (2000). Bacterial
dehalorespiration with chlorinated benzenes. Nature 408: 580–583.
Maymó-Gatell, X., Y. T. Chien, J. M. Gossett, and S. H. Zinder. (1997). Isolation
of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 276:
1568–1571.
Müller, J. A., B. M. Rosner, G. von Abendroth, G. Meshulam-Simon, P. L.
McCarty, and A. M. Spormann. (2004). Molecular identification of the catabolic
vinyl chloride reductase from Dehalococcoides sp. strain VS and its environmental
distribution. Appl. Environ. Microbiol. 70: 4880–4888.
He, J., K. M. Ritalahti, K.-L. Yang, S. S. Koenigsberg, and F. E. Löffler. (2003).
Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic
bacterium. Nature 424: 62–65.
He, J., Y. Sung, R. Krajmalnik-Brown, K. M. Ritalahti, and F. E. Löffler. (2005).
Isolation and characterization of Dehalococcoides sp. strain FL2, a trichloroethene
(TCE)- and 1,2-dichloroethene-respiring anaerobe. Environ. Microbiol. 7: 1442–1450.
Sung, Y., K. M. Ritalahti, R. P. Apkarian, and F. E. Löffler. (2006). Quantitative
PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring
Dehalococcoides isolate. Appl. Environ. Microbiol. 72: 1980–1987.
144
Literature
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
Padilla-Crespo, E., J. Yan, C. Swift, D. D. Wagner, K. Chourey, R. L. Hettich, K.
M. Ritalahti, and F. E. Löffler. (2014). Identification and environmental distribution
of dcpA, which encodes the reductive dehalogenase catalyzing the dichloroelimination
of 1,2-dichloropropane to propene in organohalide-respiring Chloroflexi. Appl.
Environ. Microbiol. 80: 808–818.
Cheng, D., and J. He. (2009). Isolation and characterization of “Dehalococcoides”
sp. strain MB, which dechlorinates tetrachloroethene to trans-1,2-dichloroethene.
Appl. Environ. Microbiol. 75: 5910–5918.
Lee, L. K., C. Ding, K. L. Yang, and J. He. (2011). Complete debromination of
tetra- and penta-brominated diphenyl ethers by a coculture consisting of
Dehalococcoides and Desulfovibrio species. Environ. Sci. Technol. 45: 8475–8482.
Duhamel, M., and E. A. Edwards. (2007). Growth and yields of dechlorinators,
acetogens, and methanogens during reductive dechlorination of chlorinated ethenes
and dihaloelimination of 1,2-dichloroethane. Environ. Sci. Technol. 41: 2303–2310.
Moe, W. M., J. Yan, M. F. Nobre, M. S. da Costa, and F. A. Rainey. (2009).
Dehalogenimonas lykanthroporepellens gen. nov., sp. nov., a reductively
dehalogenating bacterium isolated from chlorinated solvent-contaminated
groundwater. Int. J. Sys. Evol. Microbiol. 59: 2692–2697.
Bowman, K. S., M. F. Nobre, M. S. da Costa, F. A. Rainey, and W. M. Moe.
(2013). Dehalogenimonas alkenigignens sp. nov., a chlorinated-alkane-dehalogenating
bacterium isolated from groundwater. Int. J. Sys. Evol. Microbiol. 63: 1492–1498.
May, H. D., G. S. Miller, B. V. Kjellerup, and K. R. Sowers. (2008).
Dehalorespiration
with
polychlorinated
biphenyls
by
an
anaerobic
ultramicrobacterium. Appl. Environ. Microbiol. 74: 2089–2094.
Mukherjee, K., K. S. Bowman, F. A. Rainey, S. Siddaramappa, J. F.
Challacombe, and W. M. Moe. (2014). Dehalogenimonas lykanthroporepellens BLDC-9T simultaneously transcribes many rdhA genes during organohalide respiration
with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors. FEMS Microbiol. Lett.
354: 111–118.
Wu, Q., K. R. Sowers, and H. D. May. (2000). Establishment of a polychlorinated
biphenyl-dechlorinating microbial consortium, specific for doubly flanked chlorines,
in a defined, sediment-free medium. Appl. Environ. Microbiol. 66: 49–53.
Wu, Q., J. E. M. Watts, K. R. Sowers, and H. D. May. (2002). Identification of a
bacterium that specifically catalyzes the reductive dechlorination of polychlorinated
biphenyls with doubly flanked chlorines. Appl. Environ. Microbiol. 68: 807–812.
Löffler, F. E., and E. A. Edwards. (2006). Harnessing microbial activities for
environmental cleanup. Curr. Opin. Biotechnol. 17: 274–284.
Seshadri, R., L. Adrian, D. E. Fouts, J. A. Eisen, A. M. Phillippy, B. A. Methe, N.
L. Ward, W. C. Nelson, et al. (2005). Genome sequence of the PCE-dechlorinating
bacterium Dehalococcoides ethenogenes. Science 307: 105–108.
McMurdie, P. J., S. F. Behrens, J. A. Müller, J. Göke, K. M. Ritalahti, R.
Wagner, E. Goltsman, A. Lapidus, et al. (2009). Localized plasticity in the
streamlined genomes of vinyl chloride respiring Dehalococcoides. PLoS Genet. 5:
e1000714.
Kube, M., A. Beck, S. H. Zinder, H. Kuhl, R. Reinhardt, and L. Adrian. (2005).
Genome sequence of the chlorinated compound-respiring bacterium Dehalococcoides
species strain CBDB1. Nat. Biotechnol. 23: 1269–1273.
Siddaramappa, S., J. F. Challacombe, S. F. Delano, L. D. Green, H. Daligault, D.
Bruce, C. Detter, R. Tapia, et al. (2012). Complete genome sequence of
145
Literature
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
Dehalogenimonas lykanthroporepellens type strain (BL-DC-9T) and comparison to
“Dehalococcoides” strains. Stand. Genomic Sci. 6: 251–264.
Richardson, R. E. (2013). Genomic insights into organohalide respiration. Curr.
Opin. Biotechnol. 24: 498–505.
Hölscher, T., R. Krajmalnik-Brown, K. M. Ritalahti, F. von Wintzingerode, H.
Görisch, F. E. Löffler, and L. Adrian. (2004). Multiple nonidentical reductivedehalogenase-homologous genes are common in Dehalococcoides. Appl. Environ.
Microbiol. 70: 5290–5297.
Hug, L. A., F. Maphosa, D. Leys, F. E. Löffler, H. Smidt, E. A. Edwards, and L.
Adrian. (2013). Overview of organohalide-respiring bacteria and a proposal for a
classification system for reductive dehalogenases. Philos. Trans. R. Soc. Lond., B,
Biol. Sci. 368: 20120322.
Roussel, E. G., M.-A. C. Bonavita, J. Querellou, B. A. Cragg, G. Webster, D.
Prieur, and R. J. Parkes. (2008). Extending the sub-sea-floor biosphere. Science
320: 1046–1046.
Ciobanu, M.-C., G. Burgaud, A. Dufresne, A. Breuker, V. Rédou, S. B. Maamar,
F. Gaboyer, O. Vandenabeele-Trambouze, et al. (2014). Microorganisms persist at
record depths in the subseafloor of the Canterbury Basin. ISME J. 8: 1370–1380.
Kallmeyer, J., R. Pockalny, R. R. Adhikari, D. C. Smith, and S. D’Hondt. (2012).
Global distribution of microbial abundance and biomass in subseafloor sediment.
Proc. Natl. Acad. Sci. USA 109: 16213–16216.
Parkes, R. J., B. Cragg, E. Roussel, G. Webster, A. Weightman, and H. Sass.
(2014). A review of prokaryotic populations and processes in sub-seafloor sediments,
including biosphere:geosphere interactions. Mar. Geol. 352: 409–425.
D'Hondt, S., S. Rutherford, and A. J. Spivack. (2002). Metabolic activity of
subsurface life in deep-sea sediments. Science 295: 2067–2070.
Schippers, A., L. N. Neretin, J. Kallmeyer, T. G. Ferdelman, B. A. Cragg, R.
John Parkes, and B. B. Jørgensen. (2005). Prokaryotic cells of the deep sub-seafloor
biosphere identified as living bacteria. Nature 433: 861–864.
Morono, Y., T. Terada, M. Nishizawa, M. Ito, F. Hillion, N. Takahata, Y. Sano,
and F. Inagaki. (2011). Carbon and nitrogen assimilation in deep subseafloor
microbial cells. Proc. Natl. Acad. Sci. USA 108: 18295–18300.
Biddle, J. F., J. S. Lipp, M. A. Lever, K. G. Lloyd, K. B. Sørensen, R. Anderson,
H. F. Fredricks, M. Elvert, et al. (2006). Heterotrophic Archaea dominate
sedimentary subsurface ecosystems off Peru. Proc. Natl. Acad. Sci. USA 103: 3846–
3851.
Lloyd, K. G., L. Schreiber, D. G. Petersen, K. U. Kjeldsen, M. A. Lever, A. D.
Steen, R. Stepanauskas, M. Richter, et al. (2013). Predominant Archaea in marine
sediments degrade detrital proteins. Nature 496: 215–218.
Fry, J. C., R. J. Parkes, B. A. Cragg, A. J. Weightman, and G. Webster. (2008).
Prokaryotic biodiversity and activity in the deep subseafloor biosphere. FEMS
Microbiol. Ecol. 66: 181–196.
Wilms, R., B. Köpke, H. Sass, T. S. Chang, H. Cypionka, and B. Engelen. (2006).
Deep biosphere-related bacteria within the subsurface of tidal flat sediments. Environ.
Microbiol. 8: 709–719.
Blazejak, A., and A. Schippers. (2010). High abundance of JS-1- and Chloroflexirelated Bacteria in deeply buried marine sediments revealed by quantitative, real-time
PCR. FEMS Microbiol. Ecol. 72: 198–207.
146
Literature
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
Biddle, J. F., S. Fitz-Gibbon, S. C. Schuster, J. E. Brenchley, and C. H. House.
(2008). Metagenomic signatures of the Peru Margin subseafloor biosphere show a
genetically distinct environment. Proc. Natl. Acad. Sci. USA 105: 10583–10588.
Biddle, J. F., J. R. White, A. P. Teske, and C. H. House. (2011). Metagenomics of
the subsurface Brazos-Trinity Basin (IODP site 1320): comparison with other
sediment and pyrosequenced metagenomes. ISME J. 5: 1038–1047.
Webster, G., A. Blazejak, B. A. Cragg, A. Schippers, H. Sass, J. Rinna, X. Tang,
F. Mathes, et al. (2009). Subsurface microbiology and biogeochemistry of a deep,
cold-water carbonate mound from the Porcupine Seabight (IODP Expedition 307).
Environ. Microbiol. 11: 239–257.
Wasmund, K., L. Schreiber, K. G. Lloyd, D. G. Petersen, A. Schramm, R.
Stepanauskas, B. B. Jørgensen, and L. Adrian. (2014). Genome sequencing of a
single cell of the widely distributed marine subsurface Dehalococcoidia, phylum
Chloroflexi. ISME J. 8: 383–397.
Wasmund, K., C. Algora, J. Müller, M. Krüger, K. G. Lloyd, R. Reinhardt, and
L. Adrian. (2014). Development and application of primers for the class
Dehalococcoidia (phylum Chloroflexi) enables deep insights into diversity and
stratification of sub-groups in the marine subsurface. Environ. Microbiol. Doi:
10.1111/1462-2920.12510.
Kaster, A.-K., K. Mayer-Blackwell, B. Pasarelli, and A. M. Spormann. (2014).
Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of
the Peruvian Margin. ISME J. 8: 1831–1842.
Webster, G., R. John Parkes, B. A. Cragg, C. J. Newberry, A. J. Weightman, and
J. C. Fry. (2006). Prokaryotic community composition and biogeochemical processes
in deep subseafloor sediments from the Peru Margin. FEMS Microbiol. Ecol. 58: 65–
85.
Inagaki, F., T. Nunoura, S. Nakagawa, A. Teske, M. Lever, A. Lauer, M. Suzuki,
K. Takai, et al. (2006). Biogeographical distribution and diversity of microbes in
methane hydrate-bearing deep marine sediments on the Pacific Ocean Margin. Proc.
Natl. Acad. Sci. USA 103: 2815–2820.
Nunoura, T., B. Soffientino, A. Blazejak, J. Kakuta, H. Oida, A. Schippers, and
K. Takai. (2009). Subseafloor microbial communities associated with rapid turbidite
deposition in the Gulf of Mexico continental slope (IODP Expedition 308). FEMS
Microbiol. Ecol. 69: 410–424.
Kittelmann, S., and M. W. Friedrich. (2008). Novel uncultured Chloroflexi
dechlorinate perchloroethene to trans-dichloroethene in tidal flat sediments. Environ.
Microbiol. 10: 1557–1570.
Watts, J. E., S. K. Fagervold, H. D. May, and K. R. Sowers. (2005). A PCR-based
specific assay reveals a population of bacteria within the Chloroflexi associated with
the reductive dehalogenation of polychlorinated biphenyls. Microbiol. 151: 2039–
2046.
Fagervold, S. K., J. E. M. Watts, H. D. May, and K. R. Sowers. (2005). Sequential
reductive dechlorination of meta-chlorinated polychlorinated biphenyl congeners in
sediment microcosms by two different Chloroflexi phylotypes. Appl. Environ.
Microbiol. 71: 8085–8090.
Fagervold, S. K., H. D. May, and K. R. Sowers. (2007). Microbial reductive
dechlorination of Aroclor 1260 in Baltimore Harbor sediment microcosms is catalyzed
by three phylotypes within the phylum Chloroflexi. Appl. Environ. Microbiol. 73:
3009–3018.
147
Literature
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
Zanaroli, G., A. Balloi, A. Negroni, L. Borruso, D. Daffonchio, and F. Fava.
(2012). A Chloroflexi bacterium dechlorinates polychlorinated biphenyls in marine
sediments under in situ-like biogeochemical conditions. J. Haz. Materials 209: 449–
457.
Lysnes, K., I. H. Thorseth, B. O. Steinsbu, L. Øvreås, T. Torsvik, and R. B.
Pedersen. (2004). Microbial community diversity in seafloor basalt from the Arctic
spreading ridges. FEMS Microbiol. Ecol. 50: 213–230.
Parkes, R. J., B. A. Cragg, N. Banning, F. Brock, G. Webster, J. C. Fry, E.
Hornibrook, R. D. Pancost, et al. (2007). Biogeochemistry and biodiversity of
methane cycling in subsurface marine sediments (Skagerrak, Denmark). Environ.
Microbiol. 9: 1146–1161.
Wellsbury, P., K. Goodman, T. Barth, B. A. Cragg, S. P. Barnes, and R. J.
Parkes. (1997). Deep marine biosphere fuelled by increasing organic matter
availability during burial and heating. Nature 388: 573–576.
Futagami, T., Y. Morono, T. Terada, A. H. Kaksonen, and F. Inagaki. (2009).
Dehalogenation activities and distribution of reductive dehalogenase homologous
genes in marine subsurface sediments. Appl. Environ. Microbiol. 75: 6905–6909.
Kawai, M., T. Futagami, A. Toyoda, Y. Takaki, S. Nishi, S. Hori, W. Arai, T.
Tsubouchi, et al. (2014). High frequency of phylogenetically diverse reductive
dehalogenase-homologous genes in deep subseafloor sedimentary metagenomes.
Front. Microbiol. 5: 80.
Hug, L., R. Beiko, A. Rowe, R. Richardson, and E. Edwards. (2012). Comparative
metagenomics of three Dehalococcoides-containing enrichment cultures: the role of
the non-dechlorinating community. BMC Genomics 13: 327.
Hesseler, M., X. Bogdanović, A. Hidalgo, J. Berenguer, G. J. Palm, W. Hinrichs,
and U. T. Bornscheuer. (2011). Cloning, functional expression, biochemical
characterization, and structural analysis of a haloalkane dehalogenase from
Plesiocystis pacifica SIR-1. Appl. Microbiol. Biotechnol. 91: 1049–1060.
Chan, W. Y., M. Wong, J. Guthrie, A. V. Savchenko, A. F. Yakunin, E. F. Pai,
and E. A. Edwards. (2010). Sequence‐and activity‐based screening of microbial
genomes for novel dehalogenases. Microbiol. Biotechnol. 3: 107–120.
Grover, R., D. T. Waite, A. J. Cessna, W. Nicholaichuk, D. G. Irvin, L. A. Kerr,
and K. Best. (1997). Magnitude and persistence of herbicide residues in farm dugouts
and ponds in the Canadian prairies. Environ. Toxicol. Chem. 16: 638–643.
de Wit, C. A. (2002). An overview of brominated flame retardants in the
environment. Chemosphere 46: 583–624.
Jeffery S. Pettis, Elinor M. Lichtenberg, Michael Andree, Jennie Stitzinger,
Robyn Rose, and D. vanEngelsdorp. (2013). Crop pollination exposes honey bees to
pesticides which alters their susceptibility to the gut pathogen Nosema ceranae. Plos
One 8: e70182.
Birnbaum, L. (2003). Health effects of polybrominated dibenzo-p-dioxins (PBDDs)
and dibenzofurans (PBDFs). Environ. Int. 29: 855–860.
Tanabe, S., M. Watanabe, T. B. Minh, T. Kunisue, S. Nakanishi, H. Ono, and H.
Tanaka. (2003). PCDDs, PCDFs, and coplanar PCBs in albatross from the North
Pacific and Southern Oceans: levels, patterns, and toxicological implications.
Environ. Sci. Technol. 38: 403–413.
Norén, K., and D. Meironyté. (2000). Certain organochlorine and organobromine
contaminants in Swedish human milk in perspective of past 20–30 years.
Chemosphere 40: 1111–1123.
148
Literature
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
Nyholm, J. R., R. K. Asamoah, L. van der Wal, C. Danielsson, and P. L.
Andersson. (2010). Accumulation of polybrominated diphenyl ethers,
hexabromobenzene, and 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane in Earthworm
(Eisenia fetida). Effects of soil type and aging. Environ. Sci. Technol. 44: 9189–9194.
Darnerud, P. O., G. S. Eriksen, T. Jóhannesson, P. B. Larsen, and M. Viluksela.
(2001). Polybrominated diphenyl ethers: occurrence, dietary exposure, and toxicology.
Environ. Health Perspect. 109: 49.
Oaks, J. L., M. Gilbert, M. Z. Virani, R. T. Watson, C. U. Meteyer, B. A. Rideout,
H. Shivaprasad, S. Ahmed, et al. (2004). Diclofenac residues as the cause of vulture
population decline in Pakistan. Nature 427: 630–633.
Fetzner, S., and F. Lingens. (1994). Bacterial dehalogenases: biochemistry, genetics,
and biotechnological applications. Microbiol. Rev. 58: 641–685.
Ensign, S., M. Hyman, and D. Arp. (1992). Cometabolic degradation of chlorinated
alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain. Appl.
Environ. Microbiol. 58: 3038–3046.
Dabrock, B., J. Riedel, J. Bertram, and G. Gottschalk. (1992). Isopropylbenzene
(cumene) — a new substrate for the isolation of trichloroethene-degrading bacteria.
Arch. Microbiol. 158: 9–13.
Arp, D. J., C. M. Yeager, and M. R. Hyman. (2001). Molecular and cellular
fundamentals of aerobic cometabolism of trichloroethylene. Biodegradation 12: 81–
103.
Chen, K., L. Huang, C. Xu, X. Liu, J. He, S. H. Zinder, S. Li, and J. Jiang. (2013).
Molecular characterization of the enzymes involved in the degradation of a
brominated aromatic herbicide. Mol. Microbiol. 89: 1121–1139.
Utkin, I., C. Woese, and J. Wiegel. (1994). Isolation and characterization of
Desulfitobacterium dehalogenans gen. nov., sp. nov., an anaerobic bacterium which
reductively dechlorinates chlorophenolic compounds. Int. J. Syst. Bacteriol. 44: 612–
619.
Holliger, C., D. Hahn, H. Harmsen, W. Ludwig, W. Schumacher, B. Tindall, F.
Vazquez, N. Weiss, et al. (1998). Dehalobacter restrictus gen. nov. and sp. nov., a
strictly anaerobic bacterium that reductively dechlorinates tetra- and trichloroethene in
an anaerobic respiration. Arch. Microbiol. 169: 313–321.
Cole, J. R., B. Z. Fathepure, and J. M. Tiedje. (1995). Tetrachloroethene and 3chlorobenzoate dechlorination activities are co-induced in Desulfomonile tiedjei DCB1. Biodegradation 6: 167–172.
Neumann, A., H. Scholz-Muramatsu, and G. Diekert. (1994). Tetrachloroethene
metabolism of Dehalospirillum multivorans. Arch. Microbiol. 162: 295–301.
Dolfing, J., and B. K. Harrison. (1992). Gibbs free energy of formation of
halogenated aromatic compounds and their potential role as electron acceptors in
anaerobic environments. Environ. Sci. Technol. 26: 2213–2218.
Justicia-Leon, S. D., K. M. Ritalahti, E. E. Mack, and F. E. Löffler. (2012).
Dichloromethane fermentation by a Dehalobacter sp. in an enrichment culture derived
from pristine river sediment. Appl. Environ. Microbiol. 78: 1288–1291.
Kuntze, K., P. Kiefer, S. Baumann, J. Seifert, M. von Bergen, J. A. Vorholt, and
M. Boll. (2011). Enzymes involved in the anaerobic degradation of meta‐substituted
halobenzoates. Mol. Microbiol. 82: 758–769.
Holliger, C., and G. Schraa. (1994). Physiological meaning and potential for
application of reductive dechlorination by anaerobic bacteria. FEMS Microbiol. Rev.
15: 297–305.
149
Literature
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
Cole, J. R., B. Z. Fathepure, and J. M. Tiedje. (1995). Tetrachloroethene and 3chlorobenzoate dechlorination activities are co-induced in Desulfomonile tiedjei DCB1. Biodegradation 6: 167–172.
Fathepure, B. Z., and S. A. Boyd. (1988). Dependence of tetrachloroethylene
dechlorination on methanogenic substrate consumption by Methanosarcina sp. strain
DCM. Appl. Environ. Microbiol. 54: 2976–2980.
Terzenbach, D. P., and M. Blaut. (1994). Transformation of tetrachloroethylene to
trichloroethylene by homoacetogenic bacteria. FEMS Microbiol. Lett. 123: 213–218.
Holliger, C., G. Wohlfarth, and G. Diekert. (1999). Reductive dechlorination in the
energy metabolism of anaerobic bacteria. FEMS Microbiol. Rev. 22: 383–398.
Schipp, C. J., E. Marco-Urrea, A. Kublik, J. Seifert, and L. Adrian. (2013).
Organic cofactors in the metabolism of Dehalococcoides mccartyi strains. Philos.
Trans. R. Soc. Lond., B, Biol. Sci. 368: 20120321.
Bruschi, M., and F. Guerlesquin. (1988). Structure, function and evolution of
bacterial ferredoxins. FEMS Microbiol. Lett. 54: 155–175.
Miller, E., G. Wohlfarth, and G. Diekert. (1998). Purification and characterization
of the tetrachloroethene reductive dehalogenase of strain PCE-S. Arch. Microbiol.
169: 497–502.
Neumann, A., G. Wohlfarth, and G. Diekert. (1996). Purification and
characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum
multivorans. J. Biol. Chem. 271: 16515–16519.
Magnuson, J. K., M. F. Romine, D. R. Burris, and M. T. Kingsley. (2000).
Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence
of tceA and substrate range characterization. Appl. Environ. Microbiol. 66: 5141–
5147.
Maillard, J., W. Schumacher, F. Vazquez, C. Regeard, W. R. Hagen, and C.
Holliger. (2003). Characterization of the corrinoid iron-sulfur protein
tetrachloroethene reductive dehalogenase of Dehalobacter restrictus. Appl. Environ.
Microbiol. 69: 4628–4638.
Krajmalnik-Brown, R., T. Hölscher, I. N. Thomson, F. M. Saunders, K. M.
Ritalahti, and F. E. Löffler. (2004). Genetic identification of a putative vinyl
chloride reductase in Dehalococcoides sp. strain BAV1. Appl. Environ. Microbiol. 70:
6347–6351.
Suyama, A., M. Yamashita, S. Yoshino, and K. Furukawa. (2002). Molecular
characterization of the PceA reductive dehalogenase of Desulfitobacterium sp. strain
Y51. J. Bacteriol. 184: 3419–3425.
Mac Nelly, A., M. Kai, A. Svatoš, G. Diekert, and T. Schubert. (2014). Functional
heterologous production of reductive dehalogenases from Desulfitobacterium
hafniense strains. Appl. Environ. Microbiol. 80: 4313–4322.
Sjuts, H., K. Fisher, M. S. Dunstan, S. E. Rigby, and D. Leys. (2012). Heterologous
expression, purification and cofactor reconstitution of the reductive dehalogenase
PceA from Dehalobacter restrictus. Protein Expr. Purif. 85: 224–229.
Chow, W. L., D. Cheng, S. Wang, and J. He. (2010). Identification and
transcriptional analysis of trans-DCE-producing reductive dehalogenases in
Dehalococcoides species. ISME J. 4: 1020–1030.
Johnson, D. R., E. L. Brodie, A. E. Hubbard, G. L. Andersen, S. H. Zinder, and
L. Alvarez-Cohen. (2008). Temporal transcriptomic microarray analysis of
“Dehalococcoides ethenogenes” strain 195 during the transition into stationary phase.
Appl. Environ. Microbiol. 74: 2864–2872.
150
Literature
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
Wagner, A., L. Adrian, S. Kleinsteuber, J. R. Andreesen, and U. Lechner. (2009).
Transcription analysis of genes encoding homologues of reductive dehalogenases in
“Dehalococcoides” sp. strain CBDB1 by using terminal restriction fragment length
polymorphism and quantitative PCR. Appl. Environ. Microbiol. 75: 1876–1884.
Adrian, L., J. Rahnenführer, J. Gobom, and T. Hölscher. (2007). Identification of
a chlorobenzene reductive dehalogenase in Dehalococcoides sp. strain CBDB1. Appl.
Environ. Microbiol. 73: 7717–7724.
Magnuson, J. K., R. V. Stern, J. M. Gossett, S. H. Zinder, and D. R. Burris.
(1998). Reductive dechlorination of tetrachloroethene to ethene by a two-component
enzyme pathway. Appl. Environ. Microbiol. 64: 1270–1275.
Morris, R., J. Fung, B. Rahm, S. Zhang, D. Freedman, S. Zinder, and R.
Richardson. (2007). Comparative proteomics of Dehalococcoides spp. reveals strainspecific peptides associated with activity. Appl. Environ. Microbiol. 73: 320–326.
Fung, J. M., R. M. Morris, L. Adrian, and S. H. Zinder. (2007). Expression of
reductive dehalogenase genes in Dehalococcoides ethenogenes strain 195 growing on
tetrachloroethene, trichloroethene, or 2, 3-dichlorophenol. Appl. Environ. Microbiol.
73: 4439–4445.
Waller, A. S., R. Krajmalnik-Brown, F. E. Löffler, and E. A. Edwards. (2005).
Multiple reductive-dehalogenase-homologous genes are simultaneously transcribed
during dechlorination by Dehalococcoides-containing cultures. Appl. Environ.
Microbiol. 71: 8257–8264.
Christiansen, N., B. K. Ahring, G. Wohlfarth, and G. Diekert. (1998). Purification
and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase
of Desulfitobacterium hafniense. FEBS Lett. 436: 159–162.
Schumacher, W., C. Holliger, A. J. B. Zehnder, and W. R. Hagen. (1997). Redox
chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of
Dehalobacter restrictus. FEBS Lett. 409: 421–425.
Hölscher, T., H. Görisch, and L. Adrian. (2003). Reductive dehalogenation of
chlorobenzene congeners in cell extracts of Dehalococcoides sp. strain CBDB1. Appl.
Environ. Microbiol. 69: 2999–3001.
Ni, S., J. K. Fredrickson, and L. Xun. (1995). Purification and characterization of a
novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of
Desulfomonile tiedjei DCB-1. J. Bacteriol. 177: 5135–5139.
Yi, S., E. C. Seth, Y.-J. Men, S. P. Stabler, R. H. Allen, L. Alvarez-Cohen, and M.
E. Taga. (2012). Versatility in corrinoid salvaging and remodeling pathways supports
corrinoid-dependent metabolism in Dehalococcoides mccartyi. Appl. Environ.
Microbiol. 78: 7745–7752.
Kräutler, B., W. Fieber, S. Ostermann, M. Fasching, K.-H. Ongania, K. Gruber,
C. Kratky, C. Mikl, et al. (2003). The cofactor of tetrachloroethene reductive
dehalogenase of Dehalospirillum multivorans is norpseudo-B12, a new type of a
natural corrinoid. Helv. Chim. Acta. 86: 3698–3716.
Wohlfarth, G., and G. Diekert. (1997). Anaerobic dehalogenases. Curr. Opin.
Biotechnol. 8: 290–295.
Glod, G., W. Angst, C. Holliger, and R. P. Schwarzenbach. (1997). Corrinoidmediated reduction of tetrachloroethene, trichloroethene, and trichlorofluoroethene in
homogeneous aqueous solution: Reaction kinetics and reaction mechanisms. Environ.
Sci. Technol. 31: 253–260.
Payne, K. A., C. P. Quezada, K. Fisher, M. S. Dunstan, F. A. Collins, H. Sjuts, C.
Levy, S. Hay, et al. (2014). Reductive dehalogenase structure suggests a mechanism
for B12-dependent dehalogenation. Nature 517: 513–516.
151
Literature
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
Adrian, L., V. Dudková, K. Demnerová, and D. L. Bedard. (2009).
"Dehalococcoides" sp. strain CBDB1 extensively dechlorinates the commercial
polychlorinated biphenyl mixture Aroclor 1260. Appl. Environ. Microbiol. 75: 4516–
4524.
Adrian, L., S. K. Hansen, J. M. Fung, H. Görisch, and S. H. Zinder. (2007).
Growth of Dehalococcoides strains with chlorophenols as electron acceptors. Environ.
Sci. Technol. 41: 2318–2323.
Bunge, M., A. Wagner, M. Fischer, J. R. Andreesen, and U. Lechner. (2008).
Enrichment of a dioxin-dehalogenating Dehalococcoides species in two-liquid phase
cultures. Environ. Microbiol. 10: 2670–2683.
Bunge, M., L. Adrian, A. Kraus, M. Opel, W. G. Lorenz, J. R. Andreesen, H.
Görisch, and U. Lechner. (2003). Reductive dehalogenation of chlorinated dioxins
by an anaerobic bacterium. Nature 421: 357–360.
Adrian, L., and H. Görisch. (2002). Microbial transformation of chlorinated
benzenes under anaerobic conditions. Res. Microbiol. 153: 131–137.
Adrian, L., U. Szewzyk, and H. Görisch. (2000). Bacterial growth linked to
reductive dechlorination of trichlorobenzenes. Biodegradation 11: 73–81.
Jayachandran, G., H. Görisch, and L. Adrian. (2003). Dehalorespiration with
hexachlorobenzene and pentachlorobenzene by Dehalococcoides sp. strain CBDB1.
Arch. Microbiol. 180: 411–416.
Myneni, S. C. B. (2002). Formation of stable chlorinated hydrocarbons in weathering
plant material. Science 295: 1039–1041.
Krzmarzick, M. J., B. B. Crary, J. J. Harding, O. O. Oyerinde, A. C. Leri, S. C.
Myneni, and P. J. Novak. (2012). Natural niche for organohalide-respiring
Chloroflexi. Appl. Environ. Microbiol. 78: 393–401.
Keppler, F., R. Eiden, V. Niedan, J. Pracht, and H. Schöler. (2000). Halocarbons
produced by natural oxidation processes during degradation of organic matter. Nature
403: 298–301.
Carter-Franklin, J. N., and A. Butler. (2004). Vanadium bromoperoxidasecatalyzed biosynthesis of halogenated marine natural products. J. Am. Chem. Soc.
126: 15060–15066.
Ballschmiter, K. (2003). Pattern and sources of naturally produced organohalogens in
the marine environment: biogenic formation of organohalogens. Chemosphere 52:
313–324.
Schöler, H. F., and F. Keppler. (2003). Abiotic formation of organohalogens in the
terrestrial environment. Chimia 57: 33–34.
Gribble, G. W. (2003). The diversity of naturally produced organohalogens.
Chemosphere 52: 289–297.
Manefield, M., T. B. Rasmussen, M. Henzter, J. B. Andersen, P. Steinberg, S.
Kjelleberg, and M. Givskov. (2002). Halogenated furanones inhibit quorum sensing
through accelerated LuxR turnover. Microbiol. 148: 1119–1127.
Pedersén, M., P. Saenger, and L. Fries. (1974). Simple brominated phenols in red
algae. Phytochemistry 13: 2273–2279.
Ashworth, R. B., and M. J. Cormier. (1967). Isolation of 2, 6-dibromophenol from
the marine hemichordate, Balanoglossus biminiensis. Science 155: 1558–1559.
Malmvärn, A., G. Marsh, L. Kautsky, M. Athanasiadou, Å. Bergman, and L.
Asplund. (2005). Hydroxylated and methoxylated brominated diphenyl ethers in the
red algae Ceramium tenuicorne and blue mussels from the Baltic Sea. Environ. Sci.
Technol. 39: 2990–2997.
152
Literature
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
149.
150.
151.
Gribble, G. W. (1999). The diversity of naturally occurring organobromine
compounds. Chem. Soc. Rev. 28: 335–346.
La Barre, S., P. Potin, C. Leblanc, and L. Delage. (2010). The halogenated
metabolism of brown algae (Phaeophyta), its biological importance and its
environmental significance. Mar. Drugs 8: 988–1010.
Vetter, W., E. Stoll, M. J. Garson, S. J. Fahey, C. Gaus, and J. F. Müller. (2002).
Sponge halogenated natural products found at parts-per-million levels in marine
mammals. Environ. Toxicol. Chem. 21: 2014–2019.
Leri, A. C., J. A. Hakala, M. A. Marcus, A. Lanzirotti, C. M. Reddy, and S. C. B.
Myneni. (2010). Natural organobromine in marine sediments: New evidence of
biogeochemical
Br
cycling.
Glob.
Biogeochem.
Cycles
24
Doi:
10.1029/2010GB003794.
Ahn, Y.-B., S.-K. Rhee, D. E. Fennell, L. J. Kerkhof, U. Hentschel, and M. M.
Häggblom. (2003). Reductive dehalogenation of brominated phenolic compounds by
microorganisms associated with the marine sponge Aplysina aerophoba. Appl.
Environ. Microbiol. 69: 4159–4166.
Boyle, A. W., C. D. Phelps, and L. Y. Young. (1999). Isolation from estuarine
sediments of a Desulfovibrio strain which can grow on lactate coupled to the reductive
dehalogenation of 2,4,6-tribromophenol. Appl. Environ. Microbiol. 65: 1133-1140.
Fennell , D. E., S.-K. Rhee, Y.-B. Ahn, M. M. Häggblom, and L. J. Kerkhof.
(2004). Detection and characterization of a dehalogenating microorganism by terminal
restriction fragment length polymorphism fingerprinting of 16S rRNA in a
sulfidogenic, 2-bromophenol-utilizing enrichment. Appl. Environ. Microbiol. 70:
1169–1175.
He, J., K. R. Robrock, and L. Alvarez-Cohen. (2006). Microbial reductive
debromination of polybrominated diphenyl ethers (PBDEs). Environ. Sci. Technol.
40: 4429–4434.
Hug, L. A., C. J. Castelle, K. C. Wrighton, B. C. Thomas, I. Sharon, K. R.
Frischkorn, K. H. Williams, S. G. Tringe, et al. (2013). Community genomic
analyses constrain the distribution of metabolic traits across the Chloroflexi phylum
and indicate roles in sediment carbon cycling. Microbiome 1: 22.
Jørgensen, B. B. (1982). Mineralization of organic matter in the sea bed—the role of
sulphate reduction. Nature 296: 643–645.
Devereux, R., M. Delaney, F. Widdel, and D. A. Stahl. (1989). Natural relationships
among sulfate-reducing eubacteria. J. Bacteriol. 171: 6689–6695.
Barton, L. L., and F. A. Tomei. (1995). Characteristics and activities of sulfatereducing bacteria, p. 1–32. In L. L. Barton (ed.), Sulfate-reducing bacteria, vol. 8.
Springer.
Muyzer, G., and A. J. Stams. (2008). The ecology and biotechnology of sulphatereducing bacteria. Nat. Rev. Microbiol. 6: 441–454.
Schiffer, A., K. Parey, E. Warkentin, K. Diederichs, H. Huber, K. O. Stetter, P.
M. Kroneck, and U. Ermler. (2008). Structure of the dissimilatory sulfite reductase
from the hyperthermophilic Archaeon Archaeoglobus fulgidus. J. Mol. Biol. 379:
1063–1074.
Müller, A. L., K. U. Kjeldsen, T. Rattei, M. Pester, and A. Loy. (2014).
Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi) sulfite
reductases. ISME J. 9: 1152–1165.
Wagner, M., A. J. Roger, J. L. Flax, G. A. Brusseau, and D. A. Stahl. (1998).
Phylogeny of dissimilatory sulfite reductases supports an early origin of sulfate
respiration. J. Bacteriol. 180: 2975–2982.
153
Literature
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
163.
164.
165.
166.
167.
Leloup, J., H. Fossing, K. Kohls, L. Holmkvist, C. Borowski, and B. B. Jørgensen.
(2009). Sulfate-reducing bacteria in marine sediment (Aarhus Bay, Denmark):
abundance and diversity related to geochemical zonation. Environ. Microbiol. 11:
1278–1291.
Meyer, B., and J. Kuever. (2007). Phylogeny of the alpha and beta subunits of the
dissimilatory adenosine-5′-phosphosulfate (APS) reductase from sulfate-reducing
prokaryotes – origin and evolution of the dissimilatory sulfate-reduction pathway.
Microbiol. 153: 2026–2044.
Blazejak, A., and A. Schippers. (2011). Real-time PCR quantification and diversity
analysis of the functional genes aprA and dsrA of sulfate-reducing prokaryotes in
marine sediments of the Peru continental margin and the Black Sea. Front. Microbiol.
2: 253.
Moura, I., J. LeGall, A. R. Lino, H. D. Peck, G. Fauque, A. V. Xavier, D. V.
DerVartanian, J. J. G. Moura, et al. (1988). Characterization of two dissimilatory
sulfite reductases (desulforubidin and desulfoviridin) from the sulfate-reducing
bacteria. Mössbauer and EPR studies. J. Am. Chem. Soc. 110: 1075–1082.
Pereira, I. A. C., A. R. Ramos, F. Grein, M. C. Marques, S. M. Da Silva, and S. S.
Venceslau. (2011). A comparative genomic analysis of energy metabolism in sulfate
reducing bacteria and archaea. Front. Microbiol. 2: 69.
Klein, M., M. Friedrich, A. J. Roger, P. Hugenholtz, S. Fishbain, H. Abicht, L. L.
Blackall, D. A. Stahl, et al. (2001). Multiple lateral transfers of dissimilatory sulfite
reductase genes between major lineages of sulfate-reducing prokaryotes. J. Bacteriol.
183: 6028–6035.
Dhillon, A., A. Teske, J. Dillon, D. A. Stahl, and M. L. Sogin. (2003). Molecular
characterization of sulfate-reducing bacteria in the Guaymas Basin. Appl. Environ.
Microbiol. 69: 2765–2772.
Simon, J., and P. M. Kroneck. (2013). Microbial sulfite respiration. Adv. Microb.
Physiol. 62: 45–117.
Finster, K. (2008). Microbiological disproportionation of inorganic sulfur
compounds. J. Sulfur Chem. 29: 281–292.
Devkota, S., Y. Wang, M. W. Musch, V. Leone, H. Fehlner-Peach, A. Nadimpalli,
D. A. Antonopoulos, B. Jabri, et al. (2012). Dietary-fat-induced taurocholic acid
promotes pathobiont expansion and colitis in ll10-/-mice. Nature 487: 104–108.
Imachi, H., Y. Sekiguchi, Y. Kamagata, A. Loy, Y.-L. Qiu, P. Hugenholtz, N.
Kimura, M. Wagner, et al. (2006). Non-sulfate-reducing, syntrophic bacteria
affiliated with Desulfotomaculum cluster I are widely distributed in methanogenic
environments. Appl. Environ. Microbiol. 72: 2080–2091.
Schippers, A., and L. N. Neretin. (2006). Quantification of microbial communities in
near-surface and deeply buried marine sediments on the Peru continental margin using
real-time PCR. Environ. Microbiol. 8: 1251–1260.
Zehnder, A., and K. Wuhrmann. (1976). Titanium (III) citrate as a nontoxic
oxidation-reduction buffering system for the culture of obligate anaerobes. Science
194: 1165–1166.
Tschech, A., and N. Pfennig. (1984). Growth yield increase linked to caffeate
reduction in Acetobacterium woodii. Arch. Microbiol. 137: 163–167.
Lindahl, V., and L. R. Bakken. (1995). Evaluation of methods for extraction of
bacteria from soil. FEMS Microbiol. Ecol. 16: 135–142.
Amalfitano, S., and S. Fazi. (2008). Recovery and quantification of bacterial cells
associated with streambed sediments. J. Microbiol. Methods 75: 237–243.
154
Literature
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
179.
180.
181.
182.
183.
184.
185.
186.
Kallmeyer, J., D. C. Smith, A. J. Spivack, and S. D’Hondt. (2008). New cell
extraction procedure applied to deep subsurface sediments. Limnol. Oceanogr.
Methods 6: 236–245.
Neumann, A., G. Wohlfarth, and G. Diekert. (1995). Properties of tetrachloroethene
and trichloroethene dehalogenase of Dehalospirillum multivorans. Arch. Microbiol.
163: 276–281.
Hölscher, T. (2005). Biochemische und molekularbiologische Untersuchung
reduktiver dehalogenasen aus Dehalococcoides sp. Stamm CBDB1. Dissertation.
Technische Universität Berlin, Berlin.
Marco-Urrea, E., S. Paul, V. Khodaverdi, J. Seifert, M. von Bergen, U.
Kretzschmar, and L. Adrian. (2011). Identification and characterization of a Recitrate synthase in Dehalococcoides strain CBDB1. J. Bacteriol. 193: 5171–5178.
Ishihama, Y., Y. Oda, T. Tabata, T. Sato, T. Nagasu, J. Rappsilber, and M.
Mann. (2005). Exponentially modified protein abundance index (emPAI) for
estimation of absolute protein amount in proteomics by the number of sequenced
peptides per protein. Mol. Cell. Proteomics 4: 1265–1272.
Miller, D., J. Bryant, E. Madsen, and W. Ghiorse. (1999). Evaluation and
optimization of DNA extraction and purification procedures for soil and sediment
samples. Appl. Environ. Microbiol. 65: 4715–4724.
Di Giusto, D., and G. C. King. (2003). Single base extension (SBE) with
proofreading polymerases and phosphorothioate primers: improved fidelity in singlesubstrate assays. Nucleic Acids Res. 31: e7.
Tamura, K., G. Stecher, D. Peterson, A. Filipski, and S. Kumar. (2013). MEGA6:
molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725–2729.
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. (1990). Basic
local alignment search tool. J. Mol. Biol. 215: 403–410.
Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and
high throughput. Nucleic Acids Res. 32: 1792–1797.
Overbeek, R., R. Olson, G. D. Pusch, G. J. Olsen, J. J. Davis, T. Disz, R. A.
Edwards, S. Gerdes, et al. (2013). The SEED and the Rapid Annotation of microbial
genomes using Subsystems Technology (RAST). Nucleic Acids Res. 42: D206–D214.
Rutherford, K., J. Parkhill, J. Crook, T. Horsnell, P. Rice, M.-A. Rajandream,
and B. Barrell. (2000). Artemis: sequence visualization and annotation.
Bioinformatics 16: 944–945.
Tamura, K., and M. Nei. (1993). Estimation of the number of nucleotide
substitutions in the control region of mitochondrial DNA in humans and chimpanzees.
Mol. Biol. Evol. 10: 512–526.
Saitou, N., and M. Nei. (1987). The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425.
Rzhetsky, A., and M. Nei. (1993). Theoretical foundation of the minimum-evolution
method of phylogenetic inference. Mol. Biol. Evol. 10: 1073–1095.
Nei, M., and S. Kumar. (2000). Molecular evolution and phylogenetics. Oxford
University Press, New York, USA.
Letunic, I., and P. Bork. (2007). Interactive Tree Of Life (iTOL): an online tool for
phylogenetic tree display and annotation. Bioinformatics 23: 127–128.
Letunic, I., and P. Bork. (2011). Interactive Tree Of Life v2: online annotation and
display of phylogenetic trees made easy. Nucleic Acids Res.: gkr201.
Frisch, M. J., G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R.
Cheeseman, J. A. Montgomery, T. Vreven, et al. (2009). Gaussian 09, revision
D.01. Gaussian, Inc., Wallingford, CT.
155
Literature
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
197.
198.
199.
200.
201.
202.
203.
Mulliken, R. S. (1955). Electronic population analysis on LCAO-MO molecular wave
functions. I. J. Chem. Phys. 23: 1833–1840.
Hirshfeld, F. L. (1977). Bonded-atom fragments for describing molecular charge
densities. Theor. Chim. Acta 44: 129–138.
Glendening, E. D., C. R. Landis, and F. Weinhold. (2012). Natural bond orbital
methods. WIREs Comput. Mol. Sci. 2: 1–42.
Nijenhuis, I., and S. H. Zinder. (2005). Characterization of hydrogenase and
reductive dehalogenase activities of Dehalococcoides ethenogenes strain 195. Appl.
Environ. Microbiol. 71: 1664–1667.
Alaee, M., P. Arias, A. Sjödin, and Å. Bergman. (2003). An overview of
commercially used brominated flame retardants, their applications, their use patterns
in different countries/regions and possible modes of release. Environ. Int. 29: 683–
689.
Treude, T., J. Niggemann, J. Kallmeyer, P. Wintersteller, C. J. Schubert, A.
Boetius, and B. B. Jørgensen. (2005). Anaerobic oxidation of methane and sulfate
reduction along the Chilean continental margin. Geochim. Cosmochim. Acta 69:
2767–2779.
Wagner, A., M. Cooper, S. Ferdi, J. Seifert, and L. Adrian. (2012). Growth of
Dehalococcoides mccartyi strain CBDB1 by reductive dehalogenation of brominated
benzenes to benzene. Environ. Sci. Technol. 46: 8960–8968.
Marco-Urrea, E., I. Nijenhuis, and L. Adrian. (2011). Transformation and carbon
isotope fractionation of tetra- and trichloroethene to trans-dichloroethene by
Dehalococcoides sp. strain CBDB1. Environ. Sci. Technol. 45: 1555–1562.
Lu, G.-N., X.-Q. Tao, W. Huang, Z. Dang, Z. Li, and C.-Q. Liu. (2010).
Dechlorination pathways of diverse chlorinated aromatic pollutants conducted by
Dehalococcoides sp. strain CBDB1. Sci. Total Environ. 408: 2549–2554.
Reed, A. E., R. B. Weinstock, and F. Weinhold. (1985). Natural population analysis.
J. Chem. Phys. 83: 735–746.
Glendening, E. D., C. R. Landis, and F. Weinhold. (2012). Natural bond orbital
methods. WIREs Comput. Mol. Sci. 2: 1–42.
Dolfing, J., and I. Novak. (2015). The Gibbs free energy of formation of halogenated
benzenes, benzoates and phenols and their potential role as electron acceptors in
anaerobic environments. Biodegradation 26: 15–27.
Sonntag, F. (2010). Kultivierung von Dehalococcoides sp. Stamm CBDB1 in
Gegenwart verschiedener Elektronenakzeptoren und Entwicklung eines
Klonierungssystems für Dehalococcoides sp.. Studienarbeit. Technische Universität
Berlin.
Algora, C., F. Gründger, L. Adrian, V. Damm, H.-H. Richnow, and M. Krüger.
(2013). Geochemistry and Microbial Populations in Sediments of the Northern Baffin
Bay, Arctic. Geomicrobiol. J. 30: 690–705.
Pester, M., K.-H. Knorr, M. W. Friedrich, M. Wagner, and A. Loy. (2012).
Sulfate-reducing microorganisms in wetlands – fameless actors in carbon cycling and
climate change. Front. Microbiol. 3: 72.
Rinke, C., P. Schwientek, A. Sczyrba, N. N. Ivanova, I. J. Anderson, J.-F. Cheng,
A. Darling, S. Malfatti, et al. (2013). Insights into the phylogeny and coding
potential of microbial dark matter. Nature 499: 431–437.
Kjeldsen, K. U., A. Loy, T. F. Jakobsen, T. R. Thomsen, M. Wagner, and K.
Ingvorsen. (2007). Diversity of sulfate-reducing bacteria from an extreme hypersaline
sediment, Great Salt Lake (Utah). FEMS Microbiol. Ecol. 60: 287–298.
156
Literature
204.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
217.
218.
219.
220.
221.
Coolen, M. J., H. Cypionka, A. M. Sass, H. Sass, and J. Overmann. (2002).
Ongoing modification of Mediterranean Pleistocene sapropels mediated by
prokaryotes. Science 296: 2407–2410.
Fennell, D. E., I. Nijenhuis, S. F. Wilson, S. H. Zinder, and M. M. Häggblom.
(2004). Dehalococcoides ethenogenes strain 195 reductively dechlorinates diverse
chlorinated aromatic pollutants. Environ. Sci. Technol. 38: 2075–2081.
Leri, A. C., and S. C. Myneni. (2010). Organochlorine turnover in forest ecosystems:
the missing link in the terrestrial chlorine cycle. Glob. Biogeochem. Cycles 24 Doi:
10.1029/2010GB003882.
Michaelis, L., and E. S. Hill. (1933). The viologen indicators. J. Gen. Physiol. 16:
859–873.
Wardman, P. (1991). The reduction potential of benzyl viologen: an important
reference compound for oxidant/radical redox couples. Free Radical Res. 14: 57–67.
Rosenbaum, M., F. Aulenta, M. Villano, and L. T. Angenent. (2011). Cathodes as
electron donors for microbial metabolism: which extracellular electron transfer
mechanisms are involved? Bioresource Technol. 102: 324–333.
Villano, M., L. De Bonis, S. Rossetti, F. Aulenta, and M. Majone. (2011).
Bioelectrochemical hydrogen production with hydrogenophilic dechlorinating bacteria
as electrocatalytic agents. Bioresource Technol. 102: 3193–3199.
Yu, L., and M. Wolin. (1969). Hydrogenase measurement with photochemically
reduced methyl viologen. J. Bacteriol. 98: 51–55.
Aulenta, F., A. Canosa, M. Majone, S. Panero, P. Reale, and S. Rossetti. (2008).
Trichloroethene dechlorination and H2 evolution are alternative biological pathways of
electric charge utilization by a dechlorinating culture in a bioelectrochemical system.
Environ. Sci. Technol. 42: 6185–6190.
Kohn, W., A. D. Becke, and R. G. Parr. (1996). Density functional theory of
electronic structure. J. Phys. Chem. 100: 12974–12980.
Wiberg, K. B., and P. R. Rablen. (1993). Comparison of atomic charges derived via
different procedures. J. Comp. Chem. 14: 1504–1518.
Fonseca Guerra, C., J.-W. Handgraaf, E. J. Baerends, and F. M. Bickelhaupt.
(2004). Voronoi deformation density (VDD) charges: Assessment of the Mulliken,
Bader, Hirshfeld, Weinhold, and VDD methods for charge analysis. J. Comp. Chem.
25: 189–210.
Pauling, L. (1932). The nature of the chemical bond. IV. The energy of single bonds
and the relative electronegativity of atoms. J. Am. Chem. Soc. 54: 3570–3582.
Holmsgaard, P. N., A. Norman, S. C. Hede, P. H. Poulsen, W. A. Al-Soud, L. H.
Hansen, and S. J. Sørensen. (2011). Bias in bacterial diversity as a result of
Nycodenz extraction from bulk soil. Soil Biol. Biochem. 43: 2152–2159.
Boyle, A. W., M. M. Häggblom, and L. Y. Young. (1999). Dehalogenation of
lindane (γ-hexachlorocyclohexane) by anaerobic bacteria from marine sediments and
by sulfate-reducing bacteria. FEMS Microbiol. Ecol. 29: 379–387.
Tonomura, K., F. Futai, O. Tanabe, and T. Yamaoka. (1965). Defluorination of
monofluoroacetate by bacteria: part I. Isolation of bacteria and their activity of
defluorination. Agric. Biol. Chem. 29: 124–128.
Kawasaki, H., N. Tone, and K. Tonomura. (1981). Plasmid-determined
dehalogenation of haloacetates in Moraxella species. Agric. Biol. Chem. 45: 29–34.
Radianingtyas, H., G. K. Robinson, and A. T. Bull. (2003). Characterization of a
soil-derived bacterial consortium degrading 4-chloroaniline. Microbiol. 149: 32793287.
157
Literature
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
234.
235.
236.
237.
238.
Boon, N., J. Goris, P. De Vos, W. Verstraete, and E. M. Top. (2001). Genetic
diversity among 3-chloroaniline- and aniline-degrading strains of the
Comamonadaceae. Appl. Environ. Microbiol. 67: 1107-1115.
Terada, H. (1990). Uncouplers of oxidative phosphorylation. Environ. Health
Perspect. 87: 213–218.
Miyoshi, H., H. Tsujishita, N. Tokutake, and T. Fujita. (1990). Quantitative
analysis of uncoupling activity of substituted phenols with a physicochemical
substituent and molecular parameters. BBA Bioenergetics 1016: 99–106.
Escher, B. I., M. Snozzi, and R. P. Schwarzenbach. (1996). Uptake, speciation, and
uncoupling activity of substituted phenols in energy transducing membranes. Environ.
Sci. Technol. 30: 3071–3079.
Mohn, W. W., and J. M. Tiedje. (1991). Evidence for chemiosmotic coupling of
reductive dechlorination and ATP synthesis in Desulfomonile tiedjei. Arch. Microbiol.
157: 1–6.
King, G. M. (1986). Inhibition of microbial activity in marine sediments by a
bromophenol from a hemichordate. Nature 323: 257–259.
Xu, N., X. Fan, X. Yan, X. Li, R. Niu, and C. Tseng. (2003). Antibacterial
bromophenols from the marine red alga Rhodomela confervoides. Phytochemistry 62:
1221–1224.
Selent, B. (1999). Kombinierter anaerober und aerober Abbau von Chlorbenzolen mit
immobilisierten Mikroorganismen. Dissertation. Technische Universität Berlin,
Berlin.
Ramanand, K., M. Balba, and J. Duffy. (1993). Reductive dehalogenation of
chlorinated benzenes and toluenes under methanogenic conditions. Appl. Environ.
Microbiol. 59: 3266–3272.
Kuhn, E. P., G. T. Townsend, and J. M. Suflita. (1990). Effect of sulfate and
organic carbon supplements on reductive dehalogenation of chloroanilines in
anaerobic aquifer slurries. Appl. Environ. Microbiol. 56: 2630–2637.
Holtze, M. S., H. C. B. Hansen, R. K. Juhler, J. Sørensen, and J. Aamand. (2007).
Microbial degradation pathways of the herbicide dichlobenil in soils with different
history of dichlobenil-exposure. Environ. Pollution 148: 343–351.
Holtze, M. S., S. R. Sørensen, J. Sørensen, and J. Aamand. (2008). Microbial
degradation of the benzonitrile herbicides dichlobenil, bromoxynil and ioxynil in soil
and subsurface environments – insights into degradation pathways, persistent
metabolites and involved degrader organisms. Environ. Pollution 154: 155–168.
Sonnabend, A. (2011). Kultivierung von Dehalococcoides sp. Stamm CBDB1 in
Gegenwart verschiedener Elektronenakzeptoren. Studienarbeit. Technische Universität
Berlin.
Gribble, G. W. (2009). Naturally occurring organohalogen compounds – a
comprehensive update, vol. 91. Springer Science & Business Media.
Menapace, L. W., and H. G. Kuivila. (1964). Mechanism of reduction of alkyl
halides by organotin hydrides. J. Am. Chem. Soc. 86: 3047–3051.
Brown, H. C., and S. Krishnamurthy. (1969). Selective reductions. XIV. Fast
reaction of aryl bromides and iodides with lithium aluminum hydride in
tetrahydrofuran. Simple convenient procedure for the hydrogenolysis of aryl bromides
and iodides. J. Org. Chem. 34: 3918–3923.
Rahm, B. G., R. M. Morris, and R. E. Richardson. (2006). Temporal expression of
respiratory genes in an enrichment culture containing Dehalococcoides ethenogenes.
Appl. Environ. Microbiol. 72: 5486–5491.
158
Literature
239.
240.
241.
242.
243.
244.
245.
246.
247.
248.
249.
250.
251.
252.
253.
254.
255.
256.
257.
Hammett, L. P. (1937). The effect of structure upon the reactions of organic
compounds. Benzene Derivates. J. Am. Chem. Soc. 59: 96–103.
Dewick, P. M. (2006). Essentials of organic chemistry: for students of pharmacy,
medicinal chemistry and biological chemistry. John Wiley & Sons.
Banerjee, R., and S. W. Ragsdale. (2003). The many faces of vitamin B12: catalysis
by cobalamin-dependent enzymes. Ann. Rev. Biochem. 72: 209–247.
Costentin, C., M. Robert, and J.-M. Savéant. (2005). Does catalysis of reductive
dechlorination of tetra-and trichloroethylenes by vitamin B12 and corrinoid-based
dehalogenases follow an electron transfer mechanism? J. Am. Chem. Soc. 127:
12154–12155.
Pratt, D. A., and W. A. van der Donk. (2005). Theoretical investigations into the
intermediacy of chlorinated vinylcobalamins in the reductive dehalogenation of
chlorinated ethylenes. J. Am. Chem. Soc. 127: 384–396.
Pratt, D. A., and W. A. van der Donk. (2006). On the role of alkylcobalamins in the
vitamin B12-catalyzed reductive dehalogenation of perchloroethylene and
trichloroethylene. Chem. Commun. 5: 558–560.
Shey, J., and W. A. van der Donk. (2000). Mechanistic studies on the vitamin B12catalyzed dechlorination of chlorinated alkenes. J. Am. Chem. Soc. 122: 12403–
12404.
Clark, D., J. Murrell, and J. Tedder. (1963). The magnitudes and signs of the
inductive and mesomeric effects of the halogens. J. Chem. Soc.: 1250–1253.
Audsley, A., and F. R. Goss. (1942). The magnitude of the solvent effect in dipolemoment measurements. Part VI. Induced and mesomeric moments of the alkyl halides
and the halogenobenzenes. J. Chem. Soc.: 497–500 DOI: 410.1039/JR9420000497
Blanksby, S. J., and G. B. Ellison. (2003). Bond dissociation energies of organic
molecules. Acc. Chem. Res. 36: 255–263.
Julliard, M., and M. Chanon. (1983). Photoelectron-transfer catalysis: its
connections with thermal and electrochemical analogs. Chem. Rev. 83: 425–506.
Zhang, N., S. R. Samanta, B. M. Rosen, and V. Percec. (2014). Single Electron
Transfer in Radical Ion and Radical-Mediated Organic, Materials and Polymer
Synthesis. Chem. Rev. 114: 5848–5958.
Canty, A. J., and G. van Koten. (1995). Mechanisms of d8 organometallic reactions
involving electrophiles and intramolecular assistance by nucleophiles. Acc. Chem.
Res. 28: 406–413.
Epstein, L. M., and E. S. Shubina. (2002). New types of hydrogen bonding in
organometallic chemistry. Coord. Chem. Rev. 231: 165–181.
Kumar, M., and P. M. Kozlowski. (2011). A biologically relevant Co1+⋅⋅⋅ H bond:
possible implications in the protein‐induced redox tuning of Co2+/Co1+ reduction.
Angew. Chem. Int. Ed. Engl. 50: 8702–8705.
Kumar, M., N. Kumar, H. Hirao, and P. M. Kozlowski. (2012). Co2+/Co+ redox
tuning in methyltransferases induced by a conformational change at the axial ligand.
Inorg. Chem. 51: 5533–5538.
Clark, T., M. Hennemann, J. S. Murray, and P. Politzer. (2007). Halogen bonding:
the σ-hole. J. Mol. Model. 13: 291–296.
Metrangolo, P., H. Neukirch, T. Pilati, and G. Resnati. (2005). Halogen bonding
based recognition processes: a world parallel to hydrogen bonding. Acc. Chem. Res.
38: 386–395.
Metrangolo, P., and G. Resnati. (2001). Halogen bonding: a paradigm in
supramolecular chemistry. Chem. Eur. J. 7: 2511–2519.
159
Literature
258.
259.
260.
261.
262.
263.
264.
265.
266.
267.
268.
269.
270.
271.
272.
273.
274.
275.
Mukherjee, A., S. Tothadi, and G. R. Desiraju. (2014). Halogen bonds in crystal
engineering: like hydrogen bonds yet different. Acc. Chem. Res. 47: 2514–2524.
Bommer, M., C. Kunze, J. Fesseler, T. Schubert, G. Diekert, and H. Dobbek.
(2014). Structural basis for organohalide respiration. Science 346: 455–458.
Waziri, N. (2010). Die Kultivierung von anaeroben, reduktiv dechlorierenden
Mischkulturen und von Dehalococcoides sp. Stamm CBDB1 in Gegenwart
verschiedener Elektronenakzeptoren. Studienarbeit. Technische Universität Berlin.
Pöritz, M., C. L. Schiffmann, G. Hause, U. Heinemann, J. Seifert, N. Jehmlich,
M. von Bergen, I. Nijenhuis, et al. (2015). Dehalococcoides mccartyi strain DCMB5
respires a broad spectrum of chlorinated aromatic compounds. Appl. Environ.
Microbiol. 81: 587–596.
Jeschke, P., R. Nauen, M. Schindler, and A. Elbert. (2010). Overview of the status
and global strategy for neonicotinoids. J. Agric. Food Chem. 59: 2897–2908.
Dively, G. P., M. S. Embrey, A. Kamel, D. J. Hawthorne, and J. S. Pettis. (2015).
Assessment of chronic sublethal effects of imidacloprid on honey bee colony health.
PloS one 10: e0118748.
Larsen, Ø., T. Lien, and N.-K. Birkeland. (2001). A novel organization of the
dissimilatory sulfite reductase operon of Thermodesulforhabdus norvegica verified by
RT-PCR. FEMS Microbiol. Lett. 203: 81–85.
Junier, P., T. Junier, S. Podell, D. R. Sims, J. C. Detter, A. Lykidis, C. S. Han, N.
S. Wigginton, et al. (2010). The genome of the Gram‐positive metal‐and sulfate‐
reducing bacterium Desulfotomaculum reducens strain MI‐1. Environ. Microbiol. 12:
2738–2754.
Stackebrandt, E., and J. Ebers. (2006). Taxonomic parameters revisited: tarnished
gold standards. Microbiology today 33: 152–155.
Yarza, P., P. Yilmaz, E. Pruesse, F. O. Glöckner, W. Ludwig, K.-H. Schleifer, W.
B. Whitman, J. Euzéby, et al. (2014). Uniting the classification of cultured and
uncultured bacteria and archaea using 16S rRNA gene sequences. Nat. Rev.
Microbiol. 12: 635–645.
Stahl, D. A., S. Fishbain, M. Klein, B. J. Baker, and M. Wagner. (2002). Origins
and diversification of sulfate-respiring microorganisms. Antonie Van Leeuwenhoek
81: 189–195.
Perez-Jimenez, J., and L. Kerkhof. (2005). Phylogeography of sulfate-reducing
bacteria among disturbed sediments, disclosed by analysis of the dissimilatory sulfite
reductase genes (dsrAB). Appl. Environ. Microbiol. 71: 1004–1011.
Wagner, M., A. Loy, M. Klein, N. Lee, N. B. Ramsing, D. A. Stahl, and M. W.
Friedrich. (2005). Functional Marker Genes for Identification of Sulfate‐Reducing
Prokaryotes. Methods Enzymol. 397: 469–489.
Grøn, C., and B. Raben-Lange. (1992). Isolation and characterization of a
haloorganic soil humic acid. Sci. Total Environ. 113: 281–286.
Asplund, G., A. Grimvall, and C. Pettersson. (1989). Naturally produced adsorbable
organic halogens (AOX) in humic substances from soil and water. Sci. Total Environ.
81: 239–248.
Suominen, K. P., M. Liukkonen, and M. Salkinoja-Salonen. (2001). Origin of
organic halogen in boreal lakes. J. Soils Sediment 1: 2–8.
Keppler, F., and H. Biester. (2003). Peatlands: a major sink of naturally formed
organic chlorine. Chemosphere 52: 451–453.
van Pée, K.-H. (1996). Biosynthesis of halogenated metabolites by bacteria. Annu.
Rev. Microbiol. 50: 375–399.
160
Literature
276.
277.
Liu, W., H. J. Kim, E. M. Lucchetta, W. Du, and R. F. Ismagilov. (2009).
Isolation, incubation, and parallel functional testing and identification by FISH of rare
microbial single-copy cells from multi-species mixtures using the combination of
chemistrode and stochastic confinement. Lab on a Chip 9: 21532162.
Zengler, K., G. Toledo, M. Rappé, J. Elkins, E. J. Mathur, J. M. Short, and M.
Keller. (2002). Cultivating the uncultured. Proc. Natl. Acad. Sci. USA 99: 15681–
15686.
161